Wollo University

School of Bio-Science and Technology

Department of Biotechnology

Genetic Engineering (Biot.3081)

Prepared by: Muhammed S. (Ass’t Prof.)

1
Chapter 1
•Introduction to Genetic
Engineering
•History to Genetic Engineering
•Vectors in Recombinant DNA
Technology
• Plasmids
• Phages
• Cosmids
• Fosmids
• Phagmids and
• Artificial Chromosomes 2
Introduction

• Genetic Engineering / Recombinant DNA Technology

 refers to the transferring of genes from one source to
another by applying different gene transferring
mechanisms.
 However, there are several other terms that can be used
to describe the technology, including gene
manipulation, gene cloning, recombinant DNA
technology, genetic modification, and the new
genetics.
 Genetic engineering is the production of a unique DNA molecule by
joining two or more DNA fragments together not normally associated
with each other.

 It is the art of cutting and pasting genes.

 It encompasses a number of methodologies or tools to construct

new combinations of DNA (recombinant DNA or rDNA).
3
The rDNA molecule thus constructed can
be introduced into an appropriate host
cell, where it can be multiplied to
generate many copies.

=> This is known as gene cloning or

DNA cloning.

4
History of Genetic Engineering
• 1953: Watson and Crick: James Watson and Francis Crick,
discovered the structure of DNA, laying the groundwork for
genetic engineering.
• 1972: Paul Berg: Created the first recombinant DNA
molecules by combining DNA from different species,
marking the beginning of genetic engineering.
• 1973: Herbert Boyer and Stanley Cohen: Developed
techniques to cut and paste DNA using restriction enzymes
and ligases, creating the first genetically modified
organisms (GMOs) by inserting foreign DNA into bacteria.
• 1978: Human Insulin Production: Genentech announced
the successful production of human insulin in bacteria,
which was later approved by the FDA in 1982, becoming the
first genetically engineered drug.
• 1983: Polymerase Chain Reaction (PCR): Kary Mullis
developed PCR, a technique that allows for the amplification
of specific DNA sequences, revolutionizing genetic research
and diagnostics. 5
• 1986: First GM Plant: The first genetically modified plant (a
tobacco plant resistant to antibiotics) was produced.
• 1994: Flavr Savr Tomato: The first commercially grown
genetically modified food, the Flavr Savr tomato, was
approved by the FDA for human consumption.
• 1996: GM Crops: Genetically modified crops, such as
herbicide-resistant soybeans and pest-resistant cotton, were
commercialized and widely adopted in agriculture.
• 1997: Dolly the Sheep: The first mammal cloned from an
adult somatic cell using the process of nuclear transfer, Dolly
the sheep, was created by Ian Wilmut and his team at the
Roslin Institute in Scotland.
• 2000: Human Genome Project: The rough draft of the human
genome sequence was completed, providing a blueprint for
human genetics and a foundation for further genetic research.
• 2012: CRISPR-Cas9: Jennifer Doudna and Emmanuelle
Charpentier developed the CRISPR-Cas9 gene-editing
technology, allowing for precise, targeted changes to the
genome and opening new possibilities for genetic
6
engineering.
 Strong foundation of genetic engineering and
modern biotechnology was laid down by:
 Paul Berg in 1972 when he created the first
recombinant DNA molecules by combining
DNA from different species, marking the
beginning of genetic engineering.
 Cohen and Boyer in 1973 when they could
successfully introduce the desired gene of one
organism into another and clone the new gene.
 Therefore, The first gene engineers are = Boyer
and Cohen 7
The first cloning experiments
• Hamilton Smith and co-workers demonstrated
unequivocally that restriction endoncleases cleave a
specific DNA sequence. Later, Daniel Nathans used
restriction endonucleases to map the simian virus 40
(SV40) genome and to locate the origin of replication.
These major breakthroughs underscored the great
potential of restriction endonucleases for DNA work.
Building on their discoveries, the cloning experiments of
Herbert Boyer, Stanley Cohen, Paul Berg, and their
colleagues in the early 1970s ushered in the era of
recombinant DNA technology. One of the first
recombinant DNA molecules to be engineered was a
hybrid of phage λ and the SV40 mammalian DNA virus
genome. In 1974 the first eukaryotic gene was cloned.
Amplified ribosomal RNA (rRNA) genes or “ribosomal
DNA” (rDNA) from the South African clawed frog Xenopus
laevis were digested with a restriction endonuclease and
linked to a bacterial plasmid.
8
Some basic tools and techniques used for Genetic Engineering/rDNA technology

Techniques Tools
1. Isolation and purification
of nucleic acids (DNA & 1. Restriction
RNA) Enzymes
2. Construction of (Endonuclease
Restriction Map s)
3. Gene transfer
4. Gel electrophoresis
2. Vectors
5. Polymerase chain reaction 3. Ligase
(PCR) Enzymes
6. DNA sequencing 4. Host cells
7. Construction of DNA
library
5. Primers
8. Microarray 6. Probes
9. Blotting techniques 7. etc
(Southern blotting;
Northern blotting;
Western blotting, etc.)
10.etc
9
General Strategies or steps involved in genetic
engineering/rDNA technology

• Most genetic engineering experiments consist

of four major stages and one prerequisite
activity:
These are:
1. Isolation and Purification of Nucleic Acids (DNA
and RNA) (prerequisite activity)
2. Cleaving the source DNA (Enzyme restriction digest of
DNA sample and DNA plasmid vector with the same RE)
3. Making recombinants (Ligation of DNA sample
products and plasmid vector by Ligase enzyme to
produce recombinant DNA molecules)
4. Transformation of rDNA into a host cell (introduction
of vector into host cells. i.e. (e.g. E.coli cell).
5. Screening of recombinants (clones containing
10
gene of interest)
11
 12
The fundamental steps of genetic engineering
13
General strategy for
the construction of
a recombinant DNA
and its cloning. The
figure illustrates the
cloning of a human
gene in e. coli with
all the details in
each step.

14
Step 1. Isolation and Purification of Nucleic Acids
(DNA and RNA)
• The genetic engineer will, at different times, need to
prepare at least three distinct kinds of DNA.
• First, total cell DNA will often be required as a source of
material from which to obtain genes to be cloned. Total cell
DNA may be DNA from a culture of bacteria, from a plant, from
animal cells, or from any other type of organism that is being
studied. It consists of the genomic DNA of the organism
along with any additional DNA molecules, such as plasmids,
that are present.
• The second type of DNA that will be required is pure plasmid
DNA. Preparation of plasmid DNA from a culture of bacteria
follows the same basic steps as purification of total cell DNA,
with the crucial difference that at some stage the plasmid DNA
must be separated from the main bulk of chromosomal DNA
also present in the cell.
• Finally, phage DNA will be needed if a phage cloning vector is
to be used. Phage DNA is generally prepared from
bacteriophage particles rather than from infected cells, so
there is no problem with contaminating bacterial DNA.
However, special techniques are needed to remove the phage 15
Preparation of total cell DNA
• The fundamentals of DNA preparation are most easily
understood by first considering the simplest type of
DNA purification procedure, that where the entire DNA
complement of a bacterial cell is required.
• The procedure for total DNA preparation from a culture
of bacterial cells can be divided into five major stages:
1. A culture of bacteria is grown and then harvested.
2. The cells are broken open to release their
contents. (i.e. Lysis of bacterial cells)
3. This cell extract is treated to remove all
components except the DNA. i.e. Separation of
nucleic acids from other cellular components
(Recovery) and Separation of the nucleic acid (DNA
from RNA or RNA from DNA ) of interest from other
nucleic acid in a pure form (Purification)
4. Finally, concentration of the DNA or RNA.
5. Determination of purity and quantification of DNA
16
The basic steps in preparation of total cell DNA from a culture of
bacteria.

17
Cell lysis/Preparation of a cell extract
• The bacterial cell is enclosed in a cytoplasmic membrane and
surrounded by a rigid cell wall. With some species, including E.
coli, the cell wall may itself be enveloped by a second, outer
membrane. All of these barriers have to be disrupted to release
the cell components. Techniques for breaking open bacterial cells
can be divided into physical methods, in which the cells are
disrupted by mechanical forces, and chemical methods, where cell
lysis is brought about by exposure to chemical agents that affect
the integrity of the cell barriers. Chemical methods are most
commonly used with bacterial cells when the object is DNA
preparation.
• Chemical lysis generally involves one agent attacking the cell wall
and another
disrupting the cell membrane. The chemicals that are used
depend on the
species of bacterium involved, but with E. coli and related
organisms, weakening of the cell wall is usually brought about by
lysozyme, ethylenediamine tetraacetate (EDTA), or a combination
of both. Lysozyme is an enzyme that is present in egg white and in
secretions such as tears and saliva, and which digests the
polymeric compounds that give the cell wall its rigidity. EDTA
removes magnesium ions that are essential for preserving18 the
overall structure of the cell envelope, and also inhibits cellular
Having lysed the cells, the final step in preparation of a cell
extract is removal of insoluble cell debris. Components such as
partially digested cell wall fractions can be pelleted by
centrifugation, leaving the cell extract as a reasonably clear
supernatant.

Preparation of a cell extract. (a) Cell lysis. (b)

Centrifugation of the cell extract to remove
insoluble debris.
19
Recovery and Purification of DNA from a cell
extract
• In addition to DNA, a bacterial cell extract contains significant
quantities of protein and RNA. A variety of methods can be used
to recover and purify the DNA from this mixture.
• One approach is to treat the mixture with reagents which degrade
the contaminants, leaving a pure solution of DNA.
• Other methods use ion-exchange chromatography to separate
the mixture into its various components, so the DNA is removed from
the proteins and RNA in the extract.
Two approaches to
DNA purification.
(a) Treating the
mixture with
reagents which
degrade the
contaminants,
leaving a pure
solution of DNA.
(b)Separating the
mixture into
different fractions,
20
one of which is
Removing contaminants by organic extraction and
enzyme digestion
• The standard way to deproteinize a cell extract is to add
phenol or a 1 : 1 mixture of phenol and chloroform.
These organic solvents precipitate proteins but leave the
nucleic acids (DNA and RNA) in aqueous solution. The result
is that if the cell extract is mixed gently with the solvent,
and the layers then separated by centrifugation,
precipitated protein molecules are left as a white
coagulated mass at the interface between the aqueous and
organic layers. The aqueous solution of nucleic acids can
then be removed with a pipette.
• With some cell extracts the protein content is so great that a
single phenol extraction is not sufficient to completely purify the
nucleic acids. This problem could be solved by carrying out
several phenol extractions one after the other, but this is
undesirable as each mixing and centrifugation step results in a
certain amount of breakage of the DNA molecules. The answer
is to treat the cell extract with a protease such as pronase or
proteinase K before phenol extraction. These enzymes break
polypeptides down into smaller units, which are more easily
21
removed by phenol.
 Removal of protein contaminants by phenol
extraction

• Some RNA molecules, especially messenger RNA

(mRNA), are removed by phenol treatment, but
most remain with the DNA in the aqueous layer. The
only effective way to remove the RNA is with the
enzyme ribonuclease, which rapidly degrades
these molecules into ribonucleotide subunits.
22
Using ion-exchange chromatography to purify DNA from a
cell extract
• DNA and RNA are both negatively charged, as are some
proteins, and so bind to a positively charged resin. The electrical
attachment is disrupted by salt, removal of the more tightly
bound molecules requiring higher concentrations of salt. By
gradually increasing the salt concentration, different types of
molecule can be detached from the resin one after another.
• The simplest way to carry out ion-exchange chromatography is
to place the resin in a glass or plastic column and then add the
cell extract to the top. The extract passes through the column,
and because this extract contains very little salt all the
negatively charged molecules bind to the resin and are retained
in the column. If a salt solution of gradually increasing
concentration is now passed through the column, the different
types of molecule will elute (i.e., become unbound) in the
sequence protein, RNA, and finally DNA. However, such careful
separation is usually not needed so just two salt solutions are
used, one whose concentration is sufficient to elute the protein
and RNA, leaving just the DNA bound, followed by a second of a
higher concentration which elutes the DNA, now free from
protein and RNA contaminants. 23
DNA purification by ion-
exchange
chromatography.
(a) Attachment of DNA
to ion-exchange
particles.
(b) DNA is purified by
column
chromatography.
The solutions
passing through the
column can be
collected by gravity
flow or by the spin
column method, in
which the column is
placed in a low-
speed centrifuge.24
Concentration of DNA samples
• Organic extraction often results in a very thick solution of
DNA that does not need to be concentrated any further.
Other purification methods give more dilute solutions and
it is therefore important to consider methods for
increasing the DNA concentration.
• The most frequently used method of DNA concentration is
ethanol precipitation. In the presence of salt (strictly
speaking, monovalent cations such as sodium ions
(Na+)), and at a temperature of -20°C or less, absolute
ethanol efficiently precipitates polymeric nucleic acids.
With a thick solution of DNA the ethanol can be layered
on top of the sample, causing molecules to precipitate at
the interface. A spectacular trick is to push a glass rod
through the ethanol into the DNA solution. When the rod
is removed, DNA molecules adhere and can be pulled out
of the solution in the form of a long fiber. Alternatively, if
ethanol is mixed with a dilute DNA solution, the
precipitate can be collected by centrifugation, and then
redissolved in an appropriate volume of water. Ethanol 25
precipitation has the added advantage of leaving short-
Collecting DNA by ethanol precipitation. (a) Absolute ethanol is layered
on top of a
concentrated solution of DNA. Fibers of DNA can be withdrawn with a
glass rod. (b) For less concentrated solutions ethanol is added (at a 26
Other sources and methods for the preparation of
total cell DNA
• Bacteria are not the only organisms from which DNA may
be required. Total cell DNA from, for example, plants or
animals will be needed if the aim of the genetic
engineering project is to clone genes from these
organisms. Although the basic steps in DNA purification
are the same whatever the organism, some modifications
may have to be introduced to take account of the special
features of the cells being used.
• Obviously growth of cells in liquid medium is appropriate
only for bacteria, other microorganisms, and plant and
animal cell cultures. The major modifications, however, are
likely to be needed at the cell breakage stage. The
chemicals used for disrupting bacterial cells do not usually
work with other organisms: lysozyme, for example, has no
effect on plant cells. Specific degradative enzymes are
available for most cell wall types, but often physical
techniques, such as grinding frozen material with a mortar
and pestle, are more efficient. On the other hand, most
animal cells have no cell wall at all, and can be lysed 27

simply by treating with detergent.

• Another important consideration is the
biochemical content of the cells from which DNA
is being extracted. With most bacteria the main
biochemicals present in a cell extract are protein,
DNA and RNA, so phenol extraction and/or
protease treatment, followed by removal of RNA
with ribonuclease, leaves a pure DNA sample.
These treatments may not, however, be sufficient
to give pure DNA if the cells also contain
significant quantities of other biochemicals. Plant
tissues are particularly difficult in this respect as
they often contain large amounts of
carbohydrates that are not removed by phenol
extraction. Instead a different approach must be
used. One method makes use of a detergent
called cetyltrimethylammonium bromide (CTAB),
which forms an insoluble complex with nucleic
acids. 28
When CTAB is added to a plant cell extract the
nucleic acid–CTAB complex precipitates, leaving
carbohydrate, protein, and other contaminants in
the supernatant. The precipitate is then collected
by centrifugation and resuspended in 1 M sodium
chloride, which causes the complex to break down.
The nucleic acids can now be concentrated by
ethanol precipitation and the RNA removed by
ribonuclease treatment.

The CTAB method for purification of

plant DNA 29
• The need to adapt organic extraction methods to take
account of the biochemical contents of different types
of starting material has stimulated the search for DNA
purification methods that can be used with any
species. This is one of the reasons why ion-exchange
chromatography has become so popular. A similar
method involves
a compound called guanidinium thiocyanate, which
has two properties that make it useful for DNA
purification. First, it denatures and dissolves all
biochemicals other than nucleic acids and can
therefore be used to release DNA from virtually any
type of cell or tissue. Second, in the presence of
guanidinium thiocyanate, DNA binds tightly to silica
particles. This provides an easy way of recovering the
DNA from the denatured mix of biochemicals. One
possibility is to add the silica directly to the cell
extract but, as with the ion-exchange methods, it is
more convenient to use a chromatography column.
30
The silica is placed in the column and the cell extract
DNA purification by
the guanidinium
thiocyanate
and silica method. (a)
In the presence of
guanidinium thiocyanate,
DNA binds to silica
particles. (b) DNA is
purified by column
chromatography.

31
Preparation of plasmid DNA
• Purification of plasmids from a culture of bacteria
involves the same general strategy as preparation of
total cell DNA. A culture of cells, containing plasmids,
is grown in liquid medium, harvested, and a cell
extract prepared. The protein and RNA are removed,
and the DNA probably concentrated by ethanol
precipitation. However, there is an important
distinction between plasmid purification and
preparation of total cell DNA. In a plasmid preparation
it is always necessary to separate the plasmid DNA
from the large amount of bacterial chromosomal DNA
that is also present in the cells.
• Separating the two types of DNA can be very difficult,
but is nonetheless essential if the plasmids are to be
used as cloning vectors. The presence of the smallest
amount of contaminating bacterial DNA in a gene
cloning experiment can easily lead to undesirable
results. Fortunately several methods are available for
32
• The methods are based on the several physical
differences between plasmid DNA and bacterial
DNA, the most obvious of which is size. The largest
plasmids are only 8% of the size of the E. coli
chromosome, and most are much smaller than this.
Techniques that can separate small DNA molecules
from large ones should therefore effectively
purify plasmid DNA.
• In addition to size, plasmids and bacterial DNA differ
in conformation. When applied to a polymer such as
DNA, the term conformation refers to the overall
spatial configuration of the molecule, with the two
simplest conformations being linear and circular.
Plasmids and the bacterial chromosome are circular,
but during preparation of the cell extract the
chromosome is always broken to give linear
fragments. A method for separating circular from
linear molecules will therefore result in pure
plasmids. 33
1) Plasmid separation on the basis of size
• The usual stage at which size fractionation is performed is during preparation of
the cell extract. If the cells are lysed under very carefully controlled conditions,
only a minimal amount of chromosomal DNA breakage occurs. The resulting
DNA fragments are still very large—much larger than the plasmids—and can be
removed with the cell debris by centrifugation. This process is aided by the fact
that the bacterial chromosome is physically attached to the cell envelope, so
fragments of the chromosome sediment with the cell debris if these attachments
are not broken.
• Cell disruption must therefore be carried out very gently to prevent wholesale
breakage of the bacterial DNA. For E. coli and related species, controlled lysis is
performed. Treatment with EDTA and lysozyme is carried out in the
presence of sucrose, which prevents the cells from bursting immediately.
Instead,
sphaeroplasts are formed, cells with partially degraded cell walls that retain an
intact cytoplasmic membrane. Cell lysis is now induced by adding a non-ionic
detergent
such as Triton X-100 (ionic detergents, such as SDS, cause chromosomal
breakage).
This method causes very little breakage of the bacterial DNA, so centrifugation
leaves
a cleared lysate, consisting almost entirely of plasmid DNA.
• A cleared lysate will, however, invariably retain some chromosomal DNA.
Furthermore, if the plasmids themselves are large molecules, they may also
sediment with the cell debris. Size fractionation is therefore rarely sufficient on
its own, and we must
consider alternative ways of removing the bacterial DNA contaminants. 34
 35
Preparation of a cleared lysate.
2) Separation on the basis of conformation
• Before considering the ways in which conformational
differences between plasmids and bacterial DNA can be used to
separate the two types of DNA, we must look more closely at
the overall structure of plasmid DNA. It is not strictly correct to
say that plasmids have a circular conformation, because
double-stranded DNA circles can take up one of two quite
distinct configurations. Most plasmids exist in the cell as
supercoiled molecules. Supercoiling occurs because the
double helix of the plasmid DNA is partially unwound during the
plasmid replication process by enzymes called topoisomerases.
The supercoiled conformation can be maintained only if both
polynucleotide strands are intact, hence the more technical
name of covalently closed circular (ccc) DNA. If one of the
polynucleotide strands is broken the double helix reverts to its
normal relaxed state, and the plasmid takes on the alternative
conformation, called open-circular (oc). Supercoiling is
important in plasmid preparation because supercoiled
molecules can be fairly easily separated from non-supercoiled
DNA. Two different methods are commonly used. Both can
purify plasmid DNA from crude cell extracts, although in
practice best results are obtained if a cleared lysate is first
prepared. 36
Two conformations of circular double-stranded DNA: (a) supercoiled—
both strands are intact; (b) open-circular-one or both strands are
nicked.
37
a) Alkaline denaturation
• The basis of this technique is that there is a narrow pH
range at which non-supercoiled DNA is denatured,
whereas supercoiled plasmids are not. If sodium
hydroxide is added to a cell extract or cleared lysate, so
that the pH is adjusted to 12.0–12.5, then the hydrogen
bonding in non-supercoiled DNA molecules is broken,
causing the double helix to unwind and the two
polynucleotide chains to separate. If acid is now added,
these denatured bacterial DNA strands reaggregate
into a tangled mass. The insoluble network can be
pelleted by centrifugation, leaving plasmid DNA in the
supernatant. An additional advantage of this procedure
is that, under some circumstances (specifically cell lysis
by SDS and neutralization with sodium acetate), most
of the protein and RNA also becomes insoluble and can
be removed by the centrifugation step. Further
purification by organic extraction or column
chromatography may therefore not be needed if the
alkaline denaturation method is used. 38
Plasmid purification by the alkaline
denaturation method (Separation on
the basis of conformation).

39
b) Ethidium bromide–caesium chloride density
gradient centrifugation
• This is a specialized version of the more general
technique of equilibrium or density gradient
centrifugation. A density gradient is produced by
centrifuging a solution of caesium chloride (CsCl) at a
very high speed. Macromolecules present in the CsCl
solution when it is centrifuged form bands at distinct
points in the gradient. Exactly where a particular
molecule bands depends on its buoyant density:
DNA has a buoyant density of about 1.70 g/cm3, and
therefore migrates to the point in the gradient where
the CsCl density is also 1.70 g/cm3. In contrast,
protein molecules have much lower buoyant densities,
and so float at the top of the tube, whereas RNA forms
a pellet at the bottom. Density gradient centrifugation
can therefore separate DNA, RNA, and protein and is
an alternative to organic extraction or column
chromatography for DNA purification. 40
Caesium chloride density gradient centrifugation. (a) A CsCl density
gradient produced by high speed centrifugation. (b) Separation of
41
protein, DNA, and RNA in a density gradient.
• More importantly, density gradient centrifugation
in the presence of ethidium bromide (EtBr) can
be used to separate supercoiled DNA from non-
supercoiled molecules. Ethidium bromide binds to
DNA molecules by intercalating between adjacent
base pairs,
causing partial unwinding of the double helix. This
unwinding results in a decrease in the buoyant
density, by as much as 0.125 g/cm3 for linear DNA.
However, supercoiled DNA, with no free ends, has
very little freedom to unwind, and can only bind a
limited amount of EtBr. The decrease in buoyant
density of a supercoiled molecule is therefore
much less, only about 0.085 g/cm3. As a
consequence, supercoiled molecules form a band
in an EtBr–CsCl gradient at a different position to
linear and open-circular DNA.

42
Partial unwinding of the DNA double helix by EtBr intercalation between
adjacent base pairs. The normal DNA molecule shown on the left is
partially unwound by taking up four EtBr molecules, resulting in the
“stretched” structure on the right.
43
• Ethidium bromide–caesium chloride density
gradient centrifugation is a very efficient method
for obtaining pure plasmid DNA. When a cleared
lysate is subjected to this
procedure, plasmids band at a distinct point,
separated from the linear bacterial DNA, with
the protein floating on the top of the gradient
and RNA pelleted at the bottom. The position of
the DNA bands can be seen by shining
ultraviolet radiation on the tube, which causes
the bound EtBr to fluoresce. The pure plasmid
DNA is removed by puncturing the side of the
tube and withdrawing a sample with a syringe.
The EtBr bound to the plasmid DNA is extracted
with n-butanol and the CsCl removed by dialysis.
The resulting plasmid preparation is virtually
100% pure and ready for use as a cloning vector.

44
Purification of plasmid DNA by EtBr–CsCl density gradient
centrifugation.
45
Preparation of bacteriophage DNA
• The key difference between phage DNA purification and the
preparation of either total cell DNA or plasmid DNA is that for
phages the starting material is not normally a cell extract. This is
because bacteriophage particles can be obtained in large
numbers from the extracellular medium of an infected bacterial
culture. When such a culture is centrifuged, the bacteria are
pelleted, leaving the phage particles in suspension. The phage
particles are then collected from the suspension and their DNA
extracted by a single deproteinization step to remove the phage
capsid.
• This overall process is more straightforward than the procedure
used to prepare total cell or plasmid DNA. Nevertheless,
successful purification of significant quantities of phage DNA is
subject to several pitfalls. The main difficulty, especially with λ, is
growing an infected culture in such a way that the extracellular
phage titer (the number of phage particles per ml of culture) is
sufficiently high. In practical terms, the maximum titer that can
reasonably be expected for λ is 1010 per ml; yet 1010 λ particles
will yield only 500 ng of DNA. Large culture volumes, in the
range of 500–1000 ml, are therefore needed if substantial
quantities of λ DNA are to be obtained. 46
Preparation of a phage suspension from an infected
culture of bacteria.

47
Quantification of Nucleic Acids (Measurement of
DNA concentration)
• It is crucial to know exactly how much DNA is present
in a solution when carrying out a gene cloning
experiment.
• Fortunately DNA concentrations can be accurately
measured by ultraviolet (UV) absorbance
spectrophotometry. The amount of UV radiation
absorbed by a solution of DNA is directly proportional
to the amount of DNA in the sample. Usually
absorbance is measured at 260 nm, at which
wavelength an absorbance (A260) of 1.0 corresponds
to 50 mg of double-stranded DNA per ml.
Measurements of as little as 1 µl of a DNA solution can
be carried out in spectrophotometers designed
especially for this purpose.
• Ultraviolet absorbance can also be used to check the
purity of a DNA preparation. With a pure sample of 48
DNA, the ratio of the absorbances at 260 and
Storage of Nucleic Acids
• Proper storage of nucleic acids is crucial to maintain their
integrity and functionality for various molecular biology
applications. Here are some key techniques for storing
DNA and RNA:
DNA Storage Techniques:
• Temperature:
• Short-term: Store at 4°C for up to a few weeks.
• Long-term: Store at -20°C or -80°C for extended periods.
• Buffers:
• Store DNA in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to
protect against nuclease activity and degradation.
• Alternatively, DNA can be stored in nuclease-free water for some
applications, although buffering agents provide additional
stability.
• Avoiding Repeated Freeze-Thaw Cycles:
• Divide DNA into aliquots to minimize the number of freeze-thaw
cycles, which can cause shearing and degradation.
• Protection from Light:
• Store DNA in opaque containers or wrap tubes in foil to protect
49
from light, which can cause
RNA Storage Techniques:
• Temperature:
• Short-term: Store at -20°C for up to a few days.
• Long-term: Store at -80°C to prevent degradation.
• RNase-Free Environment:
• Use RNase-free tubes, tips, and reagents.
• Wear gloves and use dedicated equipment to prevent
contamination with RNases.
• Storage Buffers:
• Store RNA in RNase-free water or in specialized RNA storage
solutions such as RNAlater®.
• Aliquoting:
• Divide RNA into small aliquots to avoid repeated freeze-thaw
cycles, which can lead to degradation.
• Precipitation:
• RNA can be precipitated with ethanol or isopropanol and
stored as a pellet at -80°C. This method is often used for
long-term storage.
50
Step 2. Cleaving the source DNA
(restriction digestion of DNA sample and
plasmid DNA with the same Restriction
endonuclease)
• Once pure samples of DNA have been
prepared, the next step in a genetic
engineering experiment is construction of the
recombinant DNA molecule. To produce this
recombinant molecule, the vector, as well as
the DNA to be cloned, must be cut at specific
points and then joined together in a controlled
manner.
• The well known enzymes used for this
purpose are Restriction endonucleases and Ligases.

51
Restriction endonucleases
• also called endonuclease or Restriction
enzymes
• are Bacterial enzymes that cut DNA at
specific sites.

• How bacteria possesses these enzymes? To

protect the bacteria itself from Bacteriophage

• What kinds of bonds are broken when restriction

enzymes cut DNA?
A. Phosphodiester bonds
B. Hydrogen bonds
C. Both A and B
D. Glycosidic bonds
E. Peptide bonds 52
 - Restriction endonucleases - recognize
pallindromic sequences
- Palindrome = a word, verse, or sentence
that reads the same forwards or
backwards.

 Complete the following palindrome sequence and

name the restriction endonuclease that recognizes
this.

5' --------G ? A ? T C------ 3' 53

Why Restriction Enzymes works in
Bacteria?; how restriction enzymes
evolved in Bacteria?
• Phage (or viruses) invade all types of cells.
Bacteria are one favorite target. Defense
mechanisms have been developed by
bacteria to defend themselves from these
invasions. Bacteria have evolved a class of
enzymes that destroy foreign DNA (eg.
Virus DNA).
• i.e. to protect bacteria from bacteriophages
(Viruses).
• Infecting DNA is cleaved (restricted) by the
restriction enzyme(s) found in bacteria
preventing it from successfully replicating
and parasitizing the cell.
54
 Why the bacteria does not kill itself by its
own Restriction Enzymes? i.e. If
everything gets cleaved, how come the
bacteria does not kill itself?

Ans: Due to the restriction enzyme

modification systems
• Usually, organisms that make restriction enzymes
also make a companion modification enzyme (DNA
methyl transferase-methylase) that protects their
own DNA from cleavage. i.e. Bacteria protect their
self DNA from restriction digestion by methylation of
its recognition site. Methylation is adding a methyl
group (CH3) to DNA.
• These enzymes recognize the same DNA sequence
as the restriction enzyme they accompany, but
instead of cleaving the sequence, they disguise it by
methylating one of the bases in each DNA strand.
55
the restriction enzyme modification systems
adding a methyl group (CH3) to DNA= by DNA methyl transferase-methylase

56
Types of Restriction Endonucleases
• At present more than 2500 different
restriction endonucleases have been
identified that are isolated from a number of
species of bacteria.
• More than 300 restriction enzymes are now
available for use in the laboratory.
• These enzymes belong to three different
classes of restriction endonucleases that can
be distinguished from each other on the basis
of their structure, cofactors required and
features of their restriction and cleavage site.

57
Type I
• Type I restriction enzymes have a complex structure with three different
subunits (endonuclease, methyl transferase and recognition).
• These enzymes cut DNA at a random site, far (~1 kb) from the
recognition sequence. Their restriction sites are also complex and
discontinuous with spacers.
• They may have biological significance but are of very little practical value
since the site where the DNA is going to be digested cannot be predicted.
Type II
• Type II restriction endonucleases cleave DNA at very precise and defined
positions close to or within the recognition sequence.
• These enzymes require magnesium (Mg2+) ions as cofactors for their
activity.
• They are smaller in size, in comparison to type I and type III enzymes and
mostly bind to DNA as homodimers.
• Their recognition site is mostly symmetric (Palindrome), showing two
fold symmetry.
Type III
• This class of enzymes are large and complex proteins that cleave outside
the recognition sequence.
• The cleavage is non-specific and ~30 nucleotides away from58 the
Characteristic features of different classes of restriction enzymes

59
Nomenclature of Restriction
endonuclease
• Three letter acronym for each enzyme
derived from the source organism
First letter from genus
Next two letters represent species
• Additional letter or number represent the
strain or serotypes
• The Roman numeral represents the order
of discovery
For example. the enzyme
• HindIII was isolated from Haemophilus
influenzae serotype d. identified thirdly.
• EcoRI was isolated from Escherichia coli
serotype R. identified firstly. 60
61
Examples of Restriction Enzymes
Enzyme Organism source Recognized
Sequence

EcoRI Escherichia coli 5' GAATTC 3’

3' CTTAAG 5’

TaqI Thermus aquaticus 5' TCGA 3’

3' AGCT 5’
HindIII Haemophilus influenzae 5'AAGCTT 3’
3'TTCGAA 5’
BamHI Bacillus amyloliquefaciens 5' GGATCC 3’
3' CCTAGG 5’
AluI Arthrobacter luteus 5' AGCT 3’
3' TCGA 5’
62
The frequency of restriction
endonuclease cut sites
i.e. The frequency of recognition sequences of
restriction endonuclease in a DNA molecule
• The number of recognition sequences for a
particular restriction endonuclease in a DNA
molecule of known length can be calculated
mathematically.
• E.g. A tetranucleotide sequence (e.g., GATC)
should occur once every 44 = 256 nucleotides,
and a hexanucleotide (e.g., GGATCC) once
every 46 = 4096 nucleotides.

• Note: Restriction sites are generally not evenly spaced out

along a DNA molecule.

63
How to estimate the frequency of the
RE cut site in a DNA molecule or
genome?
Random occurrence of cut site of
endonuclease can be calculated by the
general formula as follows
Random occurrence of cut site of
endonuclease
n= number of bases

e.g. The random occurrence of the

hexameric (6) sequence:
is 1/46 =1/4096
64
65
Restriction enzymes cleave DNA in
the form of:
1) Sticky (cohesive) ends, or
2) Blunt (flush) ends
1. Sticky (cohesive) ends (i.e. when restriction
endonucleases cleave both strands of DNA at the alternate
phosphodiester bonds and leaving unpaired bases on the ends),
• Sticky (cohesive) ends are an end of a double-stranded
DNA molecule where there is a single-stranded extension. i.e.
DNA strands are cut at cleavage is staggered, so that the
resulting DNA fragments have short single-stranded overhangs
at each end.
• Sticky ends increase the efficiency of ligation. This is because
compatible sticky ends can base pair with one another by
hydrogen bonding, forming a relatively stable structure for the
enzyme to work on. If the phosphodiester bonds are not
synthesized fairly quickly then the sticky ends fall apart again.
These transient, base-paired structures do, however, increase
the efficiency of ligation by increasing the length of time the
ends are in contact with one another. 66
2. Blunt (flush) ends (i.e. when restriction
endonucleases cleave both strands of DNA at
the opposing phosphodiester bonds and
leaving no unpaired bases on the ends)

67
68
Recognition sequences and cut sites of various endonucleases

Restriction cleavage with Sma-I

resulting in blunt ends.

Blunt ends

Sticky ends

69
The restriction site sequence of EcoRI is a palindrome of a hexamer and it cuts between the bases G
and A on both the strands resulting in sticky ends or cohesive ends

70
Isoschizomers
• Different restriction enzymes sometimes
recognize and cleave within identical target
sequences. For example, MboI and Sau3A
recognize the same tetranucleotide run: 5’-
GATC-3’. Both cleave the DNA strands at the
same position, namely, on the 5’-side of the
G. Such enzymes are called isoschizomers,
meaning that they cut at the same site. The
enzyme BamHI is an isoschizomer of MboI
and Sau3A except that it has greater
specificity because it acts only at
hexanucleotide sequences reading GGATCC.
BamHI cuts between the two G’s, leaving
cohesive 5’-ends that can match up with
MboI or Sau3A fragments. 71
Construction of restriction map

Restriction map

 is a diagram that shows the location of

restriction enzyme cut sites on a segment of
DNA
 is a linear sequence of restriction sites at
defined intervals along the DNA.
 is a representing of the order and distances of
restriction endonuclease cut sites in a segment
of DNA.
• Using a restriction map to work out which
restriction endonucleases should be used to obtain
DNA fragments containing individual genes.
72
73
Construction of a restriction map

74
75
 (A) To determine the location and number of restriction
enzyme sites, a segment of DNA is digested with a
restriction enzyme. In this example, the piece of DNA is
5,000 base pairs in length. Cutting with BamHI gave three
fragments: of 3,000 bp, 1500 bp, and 500 bp. The figure
shows the three possible arrangements of these three
fragments. The fourth arrangement shown is not really
different but is merely the third possible arrangement drawn
in the opposite orientation. (B) Double digestion is the next
step in compiling a restriction map. Cutting the DNA with
EcoRI alone would give two fragments: of 4000 bp and 1000
bp. When the DNA is cut with both EcoRI and BamHI
simultaneously, four fragments are resolved by gel
electrophoresis. Two of these are identical to the 1500 bp
and 500 bp fragments from the BamHI single digest;
therefore, no EcoRI sites are present within these
fragments. The remaining two fragments, 2000 bp and 1000
bp, add up to give the 3000 bp BamHI fragment. Therefore,
the single EcoRI site must be within the 3000 bp BamHI
fragment. Of the three possible arrangements shown in part
(A), the third arrangement is ruled out (if it was cut with
76

EcoRI alone, it could not give two fragments of 4000 bp and

77
Exercise
• Determine the restriction map of a linear DNA
molecule having 1,000 base pairs long and it
is digested with the following restriction
enzymes, producing the following results:

78
Answer:
Each enzyme alone produces two fragments,
so
the molecule has one site for each enzyme Since we
get different-sized fragments with each enzyme, the
sites must be located asymmetrically along the DNA.
Draw these sites:

• The EcoRI fragment that lacks a BglII site should

appear in the double digest. If BglII cuts within the
400 base-pair fragment, we would expect to see 150,
250, and 600 base-pair fragments. We don’t see this,
so the BglII site is not within the 400 base-pair EcoRI
fragment. Thus, the map looks like this:

79
Exercise
• The linear 10-kb DNA fragment contains both
BamHI and HindIII restriction sites. Draw the
sites on the map; be specific about the
location of the sites relative to each other and
the ends of the fragment. There are two
solutions.

Digestion with Fragment sizes

(kb)
BamHI 5.0, 3.0, 2.0
HindIII 5.5, 4.5
BamHI and HindIII 5.0, 3.0, 1.5, 0.5

80
Exercise. What would be the sizes of the
fragments that you would expect to find if you cut
this plasmid with
(a) BamHI?
(b) EcoRI ?
(c) both BamHI and EcoRI together?
Ans.
a) If you cut with BamHI you
would expect two fragments of
0.5 kb and 2.8 kb.
b) If you cut with EcoRI you
would expect a single fragment
of 3.3 kb.
c) If you use both restriction
enzymes together you would
expect three fragments of 0.5,
81
0.8 and 2 kb.
82
Putting sticky ends onto a blunt-ended
molecule
• If restriction endonucleases cleave both strands of
DNA at the opposing phosphodiester bonds and
leaving no unpaired bases on the ends (blunt ends),
how can we prepare recombinant DNA (rDNA)?
• Compatible sticky ends are desirable on the DNA
molecules to be ligated together in a gene cloning
experiment. Often these sticky ends can be provided
by digesting both the vector and the DNA to be
cloned with the same restriction endonuclease, or
with different enzymes that produce the same sticky
end, but it is not always possible to do this. A
common situation is where the vector molecule has
sticky ends, but the DNA fragments to be cloned are
blunt-ended. Under these circumstances one of
three methods can be used to put the correct sticky
ends onto the DNA fragments. 83
• There are three methods that can be used to
put the correct sticky ends onto the blunt
DNA fragments.
• Using
linkers
Adaptors
homopolymer tailing

84
Generating a new RE (restriction
endonuclease) cut site at a blunt end
• Using linkers
• they are short blunt ended synthetic
dsDNA containing a RE cut site and used to
place sticky ends on to a blunt-ended DNA
molecule.
• Experimental procedure:
i. Blunt ended DNA + linker
ii. Digest with appropriate RE
iii. Ligate to vector

85
 86
Linkers and their use: (a) the structure of a typical linker; (b) the attachment of
87
• Drawback of linkers
• Consider what would happen if the blunt-ended
molecule contained one or more BamHI recognition
sequences. If this was the case, the restriction step
needed to cleave the linkers and produce the sticky
ends would also cleave the blunt-ended molecule. The
resulting fragments will have the correct sticky ends,
but that is no consolation if the gene contained in the
blunt-ended fragment has now been broken into
pieces.

A possible problem with the use of linkers. Compare this situation with
88
the desired result of BamHI restriction
Solution for this problem using
Adaptors
• The second method of attaching sticky ends
to a blunt-ended molecule is designed to
avoid this problem. Adaptors, like linkers,
are short synthetic oligonucleotides. But
unlike linkers, an adaptor is synthesized so
that it already has one sticky end.
• Drawback
• the sticky ends of individual adaptor
molecules could base pair with each
other to form dimers.

89
Adaptors and the potential problem with their use. (a) A typical
adaptor. (b) Two adaptors could ligate to one another to produce
a molecule similar to a linker, so that (c) after ligation of
adaptors a blunt-ended molecule is still blunt-ended and the 90
Solution; using DNA modifying enzymes
• Obviously, each end of a double-stranded
molecule consists of one 5′-P terminus and one 3′-
OH terminus and Ligation takes place between the
5′-P and 3′-OH ends. That is why adapter make
dimers. However, after the 5′-P terminus is
modified by alkaline phosphatase: one of the
adapter end lacks the phosphate group, and it
become a 5′-OH terminus. Hence, DNA ligase is
unable to form a phosphodiester bridge between
5′-OH and 3′-OH ends and no dimer formation.
Therefore, after the adapter is modified it can be
ligated to a blunt-ended DNA molecule but not to
themselves. After the adaptors have been
attached, the abnormal (modified) 5′-OH terminus
is converted to the natural 5′-P form by treatment
with the enzyme polynucleotide kinase,
producing a sticky-ended fragment that can be
inserted into an appropriate vector. 91
 By Alkaline
phosphatase

The use of adaptors: (a) the actual structure of an adaptor, showing the
modified 5′-OH terminus; 92
Producing sticky ends by homopolymer tailing
• The technique of homopolymer tailing offers a radically
different approach to the production of sticky ends on a
blunt-ended DNA molecule. A homopolymer is simply a
polymer in which all the subunits are the same.
• Tailing involves using the enzyme terminal
deoxynucleotidyl transferas to
add a series of nucleotides onto the 3′-OH termini of a
double-stranded DNA molecule. If this reaction is carried out
in the presence of just one deoxyribonucleotide, a
homopolymer tail is produced. Of course, to be able to ligate
together two tailed molecules, the homopolymers must be
complementary. Frequently polydeoxycytosine (poly(dC))
tails are attached to the vector and poly(dG) to the DNA to
be cloned. Base pairing between the two occurs when the
DNA molecules are mixed.
• In practice, the poly(dG) and poly(dC) tails are not usually
exactly the same length, and the base-paired recombinant
molecules that result have nicks as well as discontinuities.
Repair is therefore a two-step process, using Klenow
polymerase (DNA polymerase enzyme without nuclease 93
activity) to fill in the nicks followed by DNA ligase to
Homopolymer
tailing:
(a) synthesis of a
homopolymer tail;
(b)construction of a
recombinant DNA
molecule from a
tailed vector plus
tailed insert DNA;
(c) repair of the
recombinant DNA
molecule. 94
Remember:
once the we have the pure DNA,
construct the restriction map, and
cut the DNA with appropriate
endonuclease,
• the next step is
 selection of appropriate vector for
making rDNA and
 transferring it to the host cell
associated with the target gene
(gene ofthere
 Therefore, interest).
is a need of cloning vehicles
(vectors) and Host cells
95
Vectors (the cloning vehicles)
• are the DNA molecules, which can carry a
foreign DNA fragment to be cloned. They are
self-replicating in an appropriate host cell.
• vectors act as a vehicle for carrying foreign DNA
into a host cell for multiplication. i.e. they are
•another
There aremajor component
many possible required
choices to
of make
vector an
rDNA molecule
depending on thefor gene of
purpose cloning.
cloning and the size of
the foreign insert. The classic cloning vectors are
plasmids, phages, and cosmids, which are limited
to inserts of up to 10, 20, or 45 kb, respectively. A
new generation of artificial chromosome vectors
that can carry much larger inserts include bacterial
artificial chromosomes (BACs), yeast artificial
chromosomes (YACs), and mammalian artificial
chromosomes (MACs). 96
Based on the purpose we use Vectors can be Cloning
vectors or Expression Vectors
Cloning Vectors
• are any molecule of DNA that can replicate itself inside
a cell and is used for carrying cloned genes or
segments of DNA.
• are small pieces of DNA used for cloning and
Must
• be self replicating or integrate into the genome (i.e.
must be able to replicate within the host cell)
• be small enough to be manipulated in vitro (i.e.
ideally less than 10 kb
in size)
• have a selectable marker so transformed cells can
be isolated (e.g. antibiotic resistance gene)
• have restriction enzyme cutting sites to clone genes
into.
97
98
Expression Vectors
• In most of the cases the main purpose of the rDNA
experiment and cloning is to sufficiently multiply or
amplify the inserted DNA fragment.
• But sometimes the aim of the process will be to
produce large quantities of protein encoded by the
inserted gene. This can be accomplished by
incorporating the necessary regulatory elements
along with the gene in the vector. Such vectors
should be provided with signals necessary for
initiation and termination of transcription such as
suitable promoters and terminator sequences; and
signals for translation initiation such as a start
codon and a ribosome binding site into the vector
upstream to the multiple cloning site (MCS).
• i.e. Sometimes the goal in gene cloning is not just to replicate the
gene, but also to produce the protein that it encodes. To ensure
transcription and translation, a foreign gene is usually inserted into an
expression vector, which, in addition to the usual origin of
replication, restriction sites, and selectable markers, contains 99
sequences required for transcription and translation in bacterial cells.
Expression Vectors

• contains sequences upstream of the cloned gene that

control transcription and translation of the cloned gene.

Expression vectors
• vectors containing
the regulatory
elements and other
machinery for the
expression of the
cloned gene,
• can be used for the
production of
recombinant
proteins

100
To ensure transcription and translation, a foreign gene may be
inserted into an expression
vector—in this example, an E. coli expression vector. 101
What are Plasmids?
Plasmids
• are naturally occurring self replicating circular
extra-chromosomal double-stranded DNA
molecules found in bacterial cells.
• are found in bacterial cells but not found in
eukaryotic cells except Saccharomyces cerevisiae
(yeast), 2 µm size plasmid.
• are relatively small when compared to bacterial cell
chromosome.
• almost all the bacteria have plasmids with a low
copy number (1-4 per cell) or a high copy number
(10-100 per cell).
• although plasmids are not essential for cell
growth and division (i.e. independent genetic
elements), they often confer traits such as
antibiotic resistance on the host organism, which
102

can be a selective advantage under certain

• The most commonly used vectors in recombinant
DNA technology are:
a) Plasmid
b) Bacteriophages (λ or M13 phages)
c) Fosmids; made from plasmid component only (F-
plasmid)
d) Cosmids; Hybrid of plasmids and λ phages
e) Phagmids; Hybrid of plasmids and M13 phages
f) Bacterial artificial chromosomes (BACs)
g) Yeast artificial chromosomes (YACs)
h) Human artificial chromosomes (HACs)
i) Shuttle vectors
j) Etc. 103
104
Size and copy number
• The size and copy number of a plasmid are
particularly important as far as cloning is concerned.
• for example, plasmid size less than 10 kb is desirable for
a cloning vector. Plasmids range from about 1.0 kb for the
smallest to over 250 kb for the largest plasmids, so only a
few are useful for cloning purposes.
• The copy number refers to the number of molecules
of an individual plasmid that are normally found in a
single bacterial cell. Some plasmids, especially the
larger ones, are stringent and have a low copy
number of perhaps just one or two per cell; others,
called relaxed plasmids, are present in multiple
copies of 50 or more per cell. Generally speaking, a
useful cloning vector needs to be present in the cell
in multiple copies so that large quantities of the
recombinant DNA molecule can be obtained.
105
Classification of naturally occurring plasmids
• based on the main characteristic coded by the plasmid
genes, plasmids can be classified in to five major types:
1.Fertility or F plasmids
• carry only tra genes and have no characteristic beyond the ability to promote
conjugal transfer of plasmids. A well-known example is the F plasmid of E.
coli.
2.Resistance or R plasmids
• carry genes conferring on the host bacterium resistance to one or more
antibacterial agents, such as chloramphenicol, ampicillin, and mercury. An
example is RP4 plasmid, which is commonly found in Pseudomonas, but
also occurs in many other bacteria.
3.Col plasmids
• code for colicins, proteins that kill other bacteria. An example is ColE1
plasmid of E. coli.
4.Degradative plasmids
• allow the host bacterium to metabolize unusual molecules such as toluene
and salicylic acid, an example being TOL plasmid of Pseudomonas putida.
5.Virulence plasmids
• confer pathogenicity on the host bacterium; these include the Ti plasmid of
Agrobacterium tumefaciens, which induce crown gall disease on
dicotyledonous plants. 106
• Depending upon whether or not they carry a set of
transfer genes, called the tra genes, which promote
bacterial conjugation, plasmids can be classified in to
two major types.
1. Conjugative, or
2. Non-conjugative
• On the basis of their being maintained as multiple
copies or limited number of copies per cell, Plasmids
can also be categorized in to two major types.
1. Stringent plasmids = exist as limited number of copies
per cell
e.g. conjugative plasmids
2. Relaxed plasmids = exist as multiple copies per cell
e.g. non-conjugative plasmids

• Generally, conjugative plasmids are of relatively high

molecular weight and are present as one to three copies
per chromosome, whereas non-conjugative plasmids are
of low molecular weight and are present as multiple
copies per chromosome. 107
a) Plasmid-cloning Vectors
• For genetic engineering, naturally occurring plasmids
have been extensively modified to produce vectors that
have the desired characteristics. Over the past few
years, many different types of plasmid vectors have
been derived from the naturally occurring plasmids.
Today there are many different plasmids available for
specific purposes, often from commercial sources. i.e.
New plasmid vectors can be constructed by re-
arranging various parts of the plasmid. This can involve
addition or deletion of DNA to change the
characteristics of the vector.
• Plasmids encode only a few of the proteins required for
their own replication and in many cases encode only
one of them. All the other proteins required for
replication, e.g. DNA polymerases, DNA ligase,
helicases, etc., are provided by the host cell. Those
replication proteins that are plasmid-encoded are
located very close to the ori (origin of replication)
108
• In early cloning experiments, the cloning vectors used were
natural plasmid such as Col E1 and pSC101. While these
plasmids are small and have single sites for the common
restriction endonucleases, they have limited genetic
markers for selecting transformants. For this reason,
considerable effort was expended on constructing, in vitro,
superior cloning vectors. New plasmid vectors can be
constructed by re-arranging various parts of the plasmid.
This can involve addition or deletion of DNA to change the
characteristics of the vector. In this way a wide range of
vectors can be constructed with relative ease. The best,
and most widely used of these early purpose-built vectors
is pBR322. Plasmid pBR322 contains the ApR and TcR genes
of RSF2124 and pSC101, respectively, combined with
replication elements of pMB1, a Col E1-like plasmid.
• Plasmid-cloning vectors are derived from bacterial
plasmids and are the most widely used, versatile, and
easily manipulated ones. ColE1 of E. coli is an example of a
naturally occurring plasmid. The ori in almost all plasmid
vectors is that of ColE1.
• Example of cloning vectors derived from naturally occurring
plasmids include; pBR322, pUC8, pBR327, etc.
109
• A DNA molecule needs to display several
features to be able to act as a vector for
gene cloning.
Most importantly it must be able to
replicate within the host cell, so that
numerous copies of the recombinant DNA
molecule can be produced and passed to
the daughter cells.
A cloning vector also needs to be
relatively small, ideally less than 10 kb in
size.
A cloning vector should contain marker
gene
Two kinds of DNA molecule that satisfy
these criteria can be found in bacterial cells:
plasmids and bacteriophage chromosomes. 110
Host range of a plasmid vector
• The host range of a plasmid vector is
determined by its ori region. Plasmids whose
ori region is derived from plasmid ColE1
have a restricted host range: they only
replicate in enteric bacteria, such as E. coli,
Salmonella, etc. Other promiscuous
plasmids have a broad host range and these
include RP4 and RSF1010.

111
Nomenclature of plasmid cloning vectors
• It is a common practice to designate plasmid by a
lower case p, followed by the first letter(s) of
researcher(s) name and the numerical number given
by the workers.
• E.g., pBR322 is a plasmid discovered by Bolivar
and Rodriguez who designated it as 322.
• pBR322 is the most popular and widely common plasmid
vector in the history of gene manipulation.
• Some plasmids are given names of the places where
they are discovered
• e.g. pUC is plasmid from University of California.

Map of plasmid pBR322. Important

regions indicated are the genes for
ampicillin and tetracycline resistance
(Apr and Tcr) and the origin of
replication (ori). Some unique
restriction sites are given.
112
• The name “pBR322” conforms with the
standard rules for vector nomenclature:
• “p” indicates that this is indeed a plasmid.
• “BR” identifies the laboratory in which the vector
was originally constructed (BR stands for Bolivar
and Rodriguez, the two researchers who
developed pBR322).
• “322” distinguishes this plasmid from others
developed in the same laboratory (there are also
plasmids called pBR325, pBR327, pBR328, etc.)
A map of pBR322 showing the
positions of the ampicillin
resistance (ampR) and
tetracycline resistance (tetR)
genes, the origin of replication
(ori) and some of the most
important restriction sites.

113
The following are some of the examples of
plasmid-cloning vectors.
1) pBR322 Vectors
• This was the first designed vector for cloning purpose
• This was the first widely used, purpose built plasmid vector. It
has a number of useful features:
The useful properties of pBR322
• Its small size less than (10kb), it is important to avoid
problems such as DNA breakdown during purification.
• It carries two sets of antibiotic resistance genes (used as a
selectable marker). Either ampicillin and tetracycline
resistance gene.
• It has a reasonably high copy number (about 15 plasmids are
present in a transformed E. coli cell, but this number can be
increased,
• This indicatesup to pBR322
that, 1000–3000, by plasmid
is a very amplification
‘famous’ plasmid inthe
and has all the
presence
essential of a for
requirements protein synthesis
a cloning vector inhibitor such as
chloramphenicol)

114
Plasmid
• pBR322 was developed in the late 1970s, the first research paper
describing its use being published in 1977. Since then many other plasmid
cloning vectors have been constructed, the majority of these derived from pBR322 by
manipulations. One of the first of these was pBR327, which was produced by
removing a 1089 bp segment from pBR322. This deletion left the ampR and tetR
genes intact, but changed the replicative and conjugative abilities of the
resulting plasmid. As a result, pBR327 differs from pBR322 in two important ways:
I. pBR327 has a higher copy number than pBR322, being present at about 30–45
molecules per E. coli cell. the higher copy number of pBR327 in normal cells makes
this vector more suitable if the aim of the experiment is to study the
function of the cloned
gene. In these cases gene dosage becomes important, because the more copies
there are of a cloned gene, the more likely it is that the effect of the cloned gene on
the
host cell will be detectable. pBR327, with its high copy number, is therefore a
better choice than pBR322 for this kind of work.
II. The deletion also destroys the conjugative ability of pBR322, making pBR327
a non-conjugative plasmid that cannot direct its own transfer to other E. coli
cells. This is important for biological containment, averting the possibility of a
recombinant pBR327 molecule escaping from the test tube and colonizing bacteria
in the gut of a careless molecular biologist. In contrast, pBR322 could theoretically
be passed to natural populations of E. coli by conjugation, though in fact pBR322
also has safeguards (though less sophisticated ones) to minimize the chances of
this
happening. pBR327 is, however, preferable if the cloned gene is potentially
harmful should an accident occur.

115
2) pUC Series Vectors
• Another popular plasmid vector is the pUC series,
which is extensively used as cloning as well as
expression vector. These vectors have three
important additional features compared to
pBR322.
• High copy number.
• A mutation within the origin of replication produces
500 to 600 copies of the plasmid per cell without
amplification.
• A synthetic polylinker.
• This is a piece of man made DNA that contains
several unique restriction sites.
• Blue-white screening.
• This is a special form of insertional activation that
can be used during the primary selection of
transformants, rather than requiring a second round
of screening.
116
pUC8; a Lac selection plasmid
• pUC8 is also descended from pBR322,
although only the replication origin and the
Ampr gene remain. The nucleotide sequence of
the Ampr gene has been changed so that it no
longer contains the unique restriction sites: all
these cloning sites are now clustered into a
short segment of the lacZ′ gene carried by
pUC8.

117
• pUC8 has three important advantages that have led to it
becoming one of the most popular E. coli cloning vectors.
1. The first of these is fortuitous: the manipulations involved in construction
of pUC8 were accompanied by a chance mutation, within the origin of
replication, which results in the plasmid having a copy number of 500–
700 even before amplification. This has a significant effect on the yield of
cloned DNA obtainable from E. coli cells transformed with recombinant
pUC8 plasmids.
2. The second advantage is that identification of recombinant cells can
be achieved by a single step process, by plating onto agar
medium containing ampicillin plus X-gal. With both pBR322 and
pBR327, selection of recombinants is a two-step procedure,
requiring replica plating from one antibiotic medium to another.
A cloning experiment with pUC8 can therefore be carried out in
half the time needed with pBR322 or pBR327.
3. The third advantage of pUC8 lies with the clustering of the restriction
sites, which allows a DNA fragment with two different sticky ends (say
EcoRI at one end and BamHI at the other) to be cloned without resorting
to additional manipulations such as linker attachment. Other pUC vectors
carry different combinations of restriction sites and provide even greater
flexibility in the types of DNA fragment that can be cloned. Furthermore,
the restriction site clusters in these vectors are the same as the clusters
in the equivalent M13mp series of vectors. DNA cloned into a member of
the pUC series can therefore be transferred directly to its M13mp
counterpart, enabling the cloned gene to be obtained as single-stranded118
DNA.
b) Phage-cloning Vectors
General information about bacteriophages
• The viruses that infect bacteria are called bacteriophages. Those phages,
which most frequently infect E. coli, are used for cloning purposes.
Bacteriophages infect bacterial cells by injecting their DNA into the host
cytoplasm. This DNA in the host cell selectively replicates and expresses
the proteins required for the assembly of new phage particles. These
processes result in the production of a large number of phages,
• Bacteriophages are usually consist of a DNA genome enclosed in a protein
head (capsid). As with other viruses, they depend on the host cell for their
propagation and do not exist as free-living organisms.
• The term ‘bacteriophage’ is often shortened to ‘phage’ The plural term
‘phages’ is used when different types of phage are being considered; we
therefore talk of T4, M13, and as being phages.
• The genetic material of phage may be single- or double-stranded DNA or
RNA.
• The natural way of transferring the viral DNA into a specific bacterial host
has attracted scientists and they have modified these viral genome DNA
to use as vectors for gene cloning. Bacteriophage lambda (λ phage) and
M13 are extensively modified for the development of a phage-based
cloning vector.
• The advantages of M13-based vectors are that they contain the same
polylinker (MCS) and α-peptide fragments as the pUC series and
recombinants can be selected by the blue → white color test. 119

• The specific use of M13 is as an aid to DNA sequencing.

• Phages may be classified as either virulent or
temperate, depending on their life cycles. When a
phage enters a bacterial cell it can produce more phage
and kill the cell (this is called the lytic growth cycle), or
it can integrate into the chromosome and remain in a
quiescent state without killing the cell (this is the
lysogenic cycle). Virulent phages are those that
exhibit a lytic life cycle only. Temperate phages exhibit
lysogenic life cycles, but most can also undergo the lytic
response when conditions are suitable. The best-known
example of a temperate phage is phage λ, which has been
the subject of intense research effort and is now more or
less fully characterized in terms of its structure and
mode of action.
• The genome of phageλ is 48.5 kb in length, and encodes
some 46 genes. Its entire genome has been sequenced
(this was the first major sequencing project to be
completed, and represents one of the milestones of
molecular genetics), and all the regulatory sites are
known. About 12 bases at the ends of this λ phage 120
• Phage infection begins with adsorption, which involves the
phage
particle binding to receptors on the bacterial surface. When
the phage has adsorbed, the DNA is injected into the cell and the
life cycle can begin. The genome circularizes and the phage
initiates either the lytic or lysogenic cycle, depending on a
number of factors that include the nutritional and metabolic
state of the host cell
and the multiplicity of infection (MOI -- the ratio of phage to
bacteria during adsorption). If the lysogenic cycle is initiated, the
phage
genome integrates into the host chromosome and is maintained
as
a prophage. It is then replicated with the chromosomal DNA and
passed on to daughter cells in a stable form. If the lytic cycle is
initiated, a complex sequence of transcriptional events
essentially enables
the phage to take over the host cell and produce multiple copies
of
the genome and the structural proteins. These components are
then
assembled or packaged into mature phage, which are released
following lysis of the host cell. 121
Life cycle of bacteriophage λ. Infection occurs when a phage particle is adsorbed and
the DNA injected into the host cell. In the lytic response, the phage utilizes the host
cell replication mechanism and produces copies of the phage genome and structural
proteins. Mature phage particles are then assembled and released by lysis of the
host cell. In the lysogenic response the phage DNA integrates into the host genome122
as a prophage (P), which can be maintained through successive cell divisions. The
• To determine the number of bacteriophage present in a
suspension, serial dilutions of the phage stock are mixed with an
excess of indicator bacteria (MOI is very low) and plated onto
agar using a soft agar overlay. On incubation, the bacteria will
grow to form what is termed a bacterial lawn. Phage that grow
in this lawn will cause lysis of the cells that the phage infects,
and as this growth spreads a cleared area or plaque will
develop. Plaques can then be counted to determine the number
of plaque forming units in the stock suspension and may be
picked from the plate for further growth and analysis. Phage
may be propagated in liquid culture by infecting a growing
culture of the host cell and incubating until cell lysis is complete;
the yield of phage particles depends on the MOI and the stage in
the bacterial growth cycle at which infection occurs.

Bacteriophage plaques.
Particles of phage λ were mixed with a strain of
E. coli and plated using a soft agar overlay.
After overnight incubation the bacterial cells
grow to form a lawn, in which regions of phage
infection appear as cleared areas or plaques.
The plaques are areas where lysis of the
bacterial cells has occurred. 123
• The filamentous phage M13 differs from λ both
structurally and in its life cycle. The M13 genome is a
single-stranded circular DNA molecule 6407 bp in
length. The phage will infect only E. coli that
have F-pili (threadlike protein ‘appendages’
found on conjugation-proficient cells). When the
DNA enters the cell, it is converted to a double-
stranded molecule known as the replicative
form (RF), which replicates until there are about
100 copies in the cell. At this point DNA
replication becomes asymmetric, and single
stranded copies of the genome are produced
and extruded from the cell as M13 particles. The
bacterium is not lysed and remains viable during this
process, although growth and division are slower than
in non-infected cells.

124
• The most commonly used phages for cloning
are bacteriophage (λ phage) and
bacteriophage (phage M13).
• Phage λ vectors are particularly useful for
preparing genomic libraries, because they
can hold a larger piece of DNA than a plasmid
vector
• Placing the recombinant DNA in a phage coat
allows it to be introduced into the host
bacteria by the normal processes of phage
infection, i.e. phage adsorption followed
by DNA injection. This is known as
transduction.

125
a) Vectors based on bacteriophage λ
• The main use of all λ -based vectors is to clone DNA
fragments that are too long to be handled by plasmid
or M13 vectors. A replacement vector, such as
λEMBL4, can carry up to 20 kb of new DNA, and some
cosmids can manage fragments up to 40 kb. This
compares with a maximum insert size of about 8 kb
for most plasmids and less than 3 kb
for M13 vectors.
• The ability to clone such long DNA fragments means
that genomic libraries can be generated.

126
• The utility of phage λ as a cloning vector depends on the fact
that not all of the genome is essential for the phage to function.
Thus, there is scope for the introduction of exogenous DNA,
although certain requirements have had to be met during the
development of cloning vectors based on phage λ.
• First, the arrangement of genes on the genome will determine
which parts can be removed or replaced for the addition of
exogenous DNA. It is fortunate that the central region of the
genome (between positions 20 and 35) is largely dispensable,
so no complex rearrangement of the genome in vitro is required.
The central region controls mainly the lysogenic properties of
the phage, and much of this region can be deleted without
impairing the functions required for the lytic infection cycle.
• Second, wild-type phage will generally have multiple recognition
sites for the restriction enzymes commonly used in cloning
procedures. This can be a major problem, as it limits the choice
of sites for the insertion of DNA. In practice, it is relatively easy
to select for phage that have reduced numbers of sites for
particular restriction enzymes, and the technique of
mutagenesis in vitro may be used to modify remaining sites that
are not required. Thus, it is possible to construct phage that
have the desired combination of restriction enzyme recognition 127
sites.
• The genome of Lambda () Phage is a 48.5-kbp
linear DNA molecule that is packaged into the head
of the bacteriophage. The middle one-third of this
genome is not essential to phage infection, so λ
phage DNA has been engineered so that foreign DNA
molecules up to 16 kbp can be inserted into this
region for cloning purposes. In vitro packaging
systems are then used to package the chimeric DNA
into phage heads which, when assembled with phage
tails, form infective phage particles. Bacteria infected
with these recombinant phage produce large
numbers of phage progeny before they lyse, and
large amounts of recombinant DNA can be easily
purified from the lysate.

• The advantage with phages is that they can take up

larger DNA segments than plasmids. Hence phage
vectors are preferred for working with genomes of
human cells. 128
• One of the major drawbacks of λ vectors is that the
capsid places
a physical constraint on the amount of DNA that can be
incorporated
during phage assembly, which limits the size of exogenous DNA
fragments that can be cloned. During packaging, viable phage
particles
can be produced from DNA that is between approximately 38 and
51 kb in length. Thus, a wild-type phage genome could
accommodate only around 2.5 kb of cloned DNA before becoming
too large for
viable phage production. This limitation has been minimized by
careful construction of vectors to accept pieces of DNA that are
close to the theoretical maximum for the particular construct. Such
vectors
fall into two main classes:
1) Insertion vectors
 as the name suggests, are vectors into which DNA fragments are
inserted without removal of part of the vector DNA, and
2) Replacement or substitution vectors
 have restriction sites flanking a region of non-essential DNA that
can be removed and replaced with a DNA fragment for cloning.
This increases the size of insert that can be accepted by phage-
129

based vectors.
Insertion and replacement phage vectors. (a) An insertion
vector is shown in
part i. Such vectors have a single restriction site (RS) for cloning
into. To generate a
recombinant, the target DNA is inserted into this site. The size of
fragment that may be
cloned is therefore determined by the difference between the
vector size and the
maximum packagable fragment size (Max). Insert DNA is shaded in
130
part ii. (b) A
Insertion vectors
• Insertion vectors have a single recognition site for one or
more restriction enzymes, which enables DNA fragments to be
inserted into the λ genome. Examples of insertion vectors
include λgt10, Charon 16A and λEZAPII. Each has a single
EcoRI site into which DNA can be inserted. In λgt10 (43.3 kb)
this generates left and right ‘arms’ of 32.7 and 10.6 kb,
respectively, which can in theory accept insert DNA fragments
up to approximately 7.6 kb in length. The EcoRI site lies within
the cI gene (λ repressor), and this forms the basis of a
selection/screening method based on plaque formation and
morphology. In Charon 16A (41.8 kb), the arms generated by
EcoRI digestion are 19.9 kb (left arm) and 21.9 kb (right arm),
and fragments of up to approximately 9 kb may be cloned.
The EcoRI site in Charon 16A lies within the ꞵ-galactosidase
gene (lacZ), which enables the detection of recombinants
using X-gal.
• The three popular insertion vectors are: λgt10, Charon 16A and
λEZAPII.

• The size of the DNA fragment that an individual vector can carry depends
131 on
Bacteriophage λ insertion vectors λgt10 and
Charon 16A. The cI and lacZ genes in λgt10
and Charon 16A, respectively, are shaded.
Within these genes there is an EcoRI site for
cloning into. The lengths of the left and right
arms (LA and RA, in kb) are given. The size of
132
Replacement vectors
• Insertion vectors offer limited scope for cloning large pieces of DNA; thus,
replacement vectors were developed in which a central ‘stuffer’ fragment
is removed and replaced with the insert DNA.
• Replacement vectors are generally designed to carry larger pieces of DNA
than insertion vectors can handle.
• Replacement vectors have restriction sites flanking a region of non-
essential DNA that can be removed and replaced with a DNA fragment for
cloning. This increases the size of insert that can be accepted by phage-
based vectors.
• Two examples of λ replacement vectors are EMBL4 and Charon 40
Bacteriophage λ replacement
vectors EMBL4 and Charon 40.
The stuffer fragment in EMBL4
is 13.2 kb and is flanked by
inverted polylinkers containing
the sites for EcoRI (E), BamHI
(B), and SalI (S). In Charon 40
the polystuffer is composed of
short repeated regions that are
cleaved by NaeI. The multiple
133
cloning site (MCS) in Charon 40
Phage λ based cloning
vectors for cloning
134
inserts of large sizes.
135
b) Vectors based on bacteriophage M13
• The most essential requirement for any cloning vector is
that it has a means of replicating in the host cell. For
plasmid vectors this requirement is easy to satisfy, as
relatively short DNA sequences are able to act as plasmid
origins of replication, and most, if not all, of the enzymes
needed for replication are provided by the host cell.
Elaborate manipulations, such as those that resulted in
pBR322 are therefore possible so long as the final
construction has an intact, functional replication origin.
• With bacteriophages such as M13 and λ, the situation as
regards replication is more complex. Phage DNA molecules
generally carry several genes that are essential for
replication, including genes coding for components of the
phage protein coat and phage specific DNA replicative
enzymes. Alteration or deletion of any of these genes will
impair or destroy the replicative ability of the resulting
molecule. There is therefore much less freedom to modify
phage DNA molecules, and generally phage cloning
vectors are only slightly different from the parent 136

molecule.
• The problems in constructing a phage cloning vector are
illustrated by considering M13. The normal M13 genome is
6.4 kb in length, but most of this is taken up by ten closely
packed genes each essential for the replication of the
phage. There is only a single 507-nucleotide intergenic
sequence into which new DNA could be inserted without
disrupting one of these genes, and this region includes the
replication origin which must itself remain intact. Clearly
there is only limited scope for modifying the M13 genome.
• Two aspects of M13 infection are of value to the genetic
engineer.
• First, the Replicative form is essentially similar to a plasmid and
can be isolated and manipulated using the same techniques.
• A second advantage is that the single-stranded DNA produced
during the infection is useful in techniques such as DNA
sequencing by the dideoxy method. This aspect alone made
M13 immediately attractive as a potential vector.
• Unlike plasmids phage M13 is single-stranded DNA
molecule. This is very important for DNA
sequencing purpose. Particularly, the Sanger method
of sequencing which requires single-stranded DNA as the
starting material. 137
• The advantages of M13 include its efficiency in
producing single-stranded DNA for sequencing and
site-directed mutagenesis, as well as its minimal
disruption to the host cell growth.

• The first step in construction of an M13 cloning vector

was to introduce the lacZ′ gene into the intergenic
sequence. This gave rise to M13mp1, which forms blue
plaques on X-gal agar. M13mp1 does not possess any
unique restriction sites in the lacZ′ gene. It does,
however, contain the hexanucleotide GGATTC near the
start of the gene. A single nucleotide change would
make this GAATTC, which is an EcoRI site. This
alteration was carried out using in vitro mutagenesis,
resulting in M13mp2. M13mp2 has a slightly altered
lacZ′ gene (the sixth codon now specifies asparagine
instead of aspartic acid), but the ꞵ-galactosidase
enzyme produced by cells infected with M13mp2 is
still perfectly functional. 138
The M13
genome,
showing
the
positions of
genes I to
X.

Construction of (a) M13mp1, and (b) M13mp2 from

the wild-type M13 genome 139
• The next step in the development of M13 vectors was
to introduce additional restriction sites into the lacZ′
gene. This was achieved by synthesizing in the test
tube a short oligonucleotide, called a polylinker,
which consists of a series of restriction sites and has
EcoRI sticky ends. This polylinker was inserted into
the EcoRI site of M13mp2, to give M13mp7, a more
complex vector with four possible cloning sites
(EcoRI, BamHI, SalI, and PstI). The polylinker is
designed so that it does not totally disrupt the lacZ′
gene: a reading frame is maintained throughout the
polylinker, and a functional, though altered, ꞵ-
galactosidase enzyme is still produced. The most
sophisticated M13 vectors have more complex
polylinkers inserted into the lacZ′ gene. An example
is M13mp8, which has the same series of restriction
sites as the plasmid pUC8. As with the plasmid
vector, one advantage of M13mp8 is its ability to
take DNA fragments with two different sticky ends. 140
Construction of M13mp7: (a) the polylinker, and (b) its insertion into the
EcoRI site of M13mp2
141
Other vectors
• In the 1970s, when recombinant DNA technology was first being
developed, only a limited number of vectors were available and these
were based on either high-copy-number plasmids or phage λ. Later,
phage M13 was developed as a specialist vector to facilitate DNA
sequencing. Gradually, a number of purpose-built vectors were
developed, of which pBR322 is probably the best example, but the
creation of each one was a major task. Over time, a series of specialist
vectors was constructed, each for a particular purpose.
• So far we have concentrated on what we might call ‘basic’ plasmid and
bacteriophage vectors for use in E. coli hosts. Although these vectors still
represent a major part of the technology of gene manipulation, there has
been continued development of more sophisticated bacterial vectors, as
well as vectors for other organisms. One driving force in this has been the
need to clone and analyze ever larger pieces of DNA, as the emphasis in
molecular biology has shifted towards the analysis of genomes rather
than simply genes in isolation. In addition, the commercial development
of integrated approaches to cloning procedures has required new vectors.
These includes
• Fosmids
• Cosmids (hybrid vector)
• Phagmids (hybrid vector)
• Artificial chromosome vectors
• Shuttle vectors 142
• etc
Fosmids
• are derived from F-plasmid, ensuring low-copy-
number replication and stable maintenance in
host cells without using phage packaging.
• They do not contain any phage-derived
components
• are considered a type of bacterial artificial
chromosome (BAC) vector, allowing them to
maintain larger DNA inserts more stably.
• are commonly used for genomic library
construction and genome sequencing projects.
• have high structural stability and have been
found to maintain human DNA effectively even
after 100 generations of bacterial growth.

143
• Phagmids/Hybrid vector combining plasmid and M13 phage.
• Although M13 vectors are very useful for the production of
single-stranded versions of cloned genes, they do suffer from
one disadvantage. There is a limit to the size of DNA fragment
that can be cloned with an M13 vector, with 1500 bp generally
being looked on as the maximum capacity, though fragments
up to 3 kb have occasionally been cloned. To get around this
problem a number of hybrid vectors (“phagemids”) have been
developed by combining a part of the M13 genome with
plasmid DNA.
• Therefore, Phagmids are a plasmid vector that contains an M13
origin of replication and which can therefore be obtained as
both double- and single-stranded DNA versions. Phagmids
avoid the instabilities of M13 cloning and can be used with
fragments up to 10 kb or more.
• i.e. The association of plasmids and M13 ori region = Phagmids
• Phage-derived origin of replication for packaging into phage
particles
• They possess multiple cloning sites and an inducible lac gene
promoter.
• They are identified by blue-white screening.
• An example of Phagmid vector is pEMBL8, which was made 144
by
transferring into pUC8 a 1300 bp fragment of the M13 genome.
Cosmid-cloning Vectors
• The final and most sophisticated type of λ-based vector
• are the vectors possessing the characteristics of both
plasmid and λ bacteriophage.
• They can be constructed by adding a fragment of
phage λ DNA including cos site, to plasmids. i.e.
Cosmids are hybrids between a phage DNA molecule and a
bacterial plasmid (a cosmid is basically a plasmid that carries a
cos site).
• Therefore, vectors that are made up of plasmid sequences joined
to the
cos sites of phage λ are known as cosmids. i.e a plasmid that
contains a cos site from the lambda phage genome.
• In this vector a large foreign DNA can be inserted.
• i.e. Long DNA fragments can be cloned using a cosmid
• The recombinant DNA so formed can be packed as phages and
injected into E.coli. Once inside the host cell, cosmids behave just
like plasmids and replicate. The advantage with cosmids is that
they can carry larger fragments of foreign DNA compared to
plasmids. Cosmids can accept DNA inserts of around 35-45 kb. 145
• With a cosmid vector of 5 kb, we demand the
insertion of 32–47 kb of foreign DNA – much
more than a phage-λ vector can
accommodate. Cosmids provide an efficient
means of cloning large pieces of foreign DNA.
Therefore, cosmid considered under Vectors
for cloning large fragments of DNA.
Because of their capacity for large fragments
of DNA, cosmids are particularly attractive
vectors for constructing libraries of
146

eukaryotic genome fragments.

• Cosmid-cloning vectors were among the first
large insert cloning vehicles developed. They
were constructed by certain features of
plasmid and the cos site of λ phage which is
required for packaging DNA into phage
particles. Cosmid replicates as a plasmid (it
contains a ColE1 origin of replication), and
uses Ampr for positive selection and employs
lambda phage packaging to select for
recombinant plasmids carrying foreign DNA
inserts up to 45 kb in size.
• The simplest cosmid vector has a ColE1
origin of replication, selectable markers
including the antibiotic-resistance gene and
β-galactosidase gene (a part of lac Z gene),
and suitable polylinker sites and λ cos site.
147
 d. Artificial chromosome vectors
• Bacterial artificial chromosomes (BACs) and yeast artificial
chromosomes (YACs) are important tools for mapping and analysis of
complex eukaryotic genomes. Much of the work on the Human Genome
Project and other genome sequencing projects depends on the use of
BACs and YACs, because they can hold greater than 300 kb of foreign
DNA.
i) Bacterial artificial chromosomes (BACs)
• BACs are constructed using the fertility factor plasmid (F factor)
of E. coli as a starting point which is larger than the other
plasmids used as cloning vectors. BACs are vectors originally
constructed from the F plasmid (a special plasmid that controls
mating and the transfer of genetic material in some bacteria The
plasmid is naturally 100 kb in size and occurs at a very low copy
number in the host. The engineered BAC vector is 7.4 kb
(including a replication origin, cloning sites, and selectable
markers) and thus can accommodate a large insert of foreign
DNA.
• F-plasmid BACs can accept DNA inserts of around 200-500 kb.
ii) Yeast artificial chromosomes (YACs)
• is a synthetic DNA that can accept large fragments of foreign
DNA (particularly human DNA). It is thus possible to clone large
• Both YACs
DNA and by
pieces BACs were
using very
YAC. useful
YACs in genome-sequencing
can accept DNA inserts of around
148
projects such as
200-2000 kb.
iii) Human artificial chromosome (HAC)
• is a synthetically produced vector DNA,
possessing the characteristics of human
chromosome. HAC may be considered as a
self-replicating micro-chromosome with a
size ranging from 1/10th to 1/5th of a human
chromosome.
• The advantage with HAC is that
• it can carry human genes that are too long.
• Further, HAC can carry genes to be introduced
into the cells in gene therapy.

149
150
151
Shuttle Vectors
• Shuttle vectors are plasmids capable of propagating and
transferring (“shuttling”) genes between two different organisms,
one of which is typically a prokaryote (E. coli) and the other a
eukaryote (for example, yeast).
• Shuttle vectors have the advantage that eukaryotic genes can be
cloned in bacterial hosts.

A typical shuttle vector. This vector has both yeast and bacterial origins
of replication, ampr (ampicillin resistance gene for selection in E. coli)
and LEU2, a gene in the yeast pathway for leucine biosynthesis. The
recipient yeast cells are LEU2 (defective in this gene) and thus require
152
leucine for growth. LEU2 yeast cells transformed with this shuttle vector
3. Making recombinant DNA (rDNA)
• After the appropriate vector and the target gene have
been selected and cleaved the next step is making
rDNA. This is accomplished by Using DNA Ligase
Enzymes- a protein molecules used to put together DNA (i.e.
catalyze the formation of a phosphodiester bond between
adjacent 3’-OH and 5’-P group in DNA).
DNA Ligase
This enzyme is responsible for the formation of the
phosphodiester linkage between two adjacent
nucleotides and thus joins two double-stranded DNA
fragments. The phosphodiester linkage is formed
between the 3′OH group (hydroxyl group at the 3′
carbon) and 5′ PO4 group of the pentose sugar of
adjacent nucleotides of two different DNA fragments or
nicks created in a double-stranded DNA molecule. In
rDNA experiments, DNA ligase is used to join two
different DNA fragments (vector and the foreign DNA) 153

that are annealed by the sticky ends. There are

Action of DNA ligase (phage T4 DNA ligase)
Two polynucleotide chains, one with a free 3-OH group
and one with a free 5-phosphate group, are joined by
DNA ligase, which forms a phosphodiester bond.

Ligation: the final step in construction of a

recombinant DNAwhy
Q. In a DNA ligation, molecule
is it important to inactivate the restriction
enzymes before adding the DNA ligase?
Ans. If this is not done, the enzymes will continue to recut the DNA
every time the DNA ligase seals the gap. 154
155
Self-ligation problem during rDNA
experiments and its solution
• In rDNA experiments there is always the problem of
self-ligation and the reformation of the original plasmid
or the vector. This reduces the efficiency of the
recombinant DNA formation. Self-ligation is the process
of annealing the sticky ends of the linearized vector
without inserting the foreign DNA fragment. On ligation
it will result in the original vector without any insert
DNA. This can be prevented by using alkaline
phosphatase. We know that the presence of 3′OH
group and 5′ PO4 group is a prerequisite for the ligase
enzyme to act. If any one group of this is absent, the
DNA ligase cannot function. Alkaline phosphatase can
remove the phosphate group from the 5′ end of the
DNA molecule resulting in a 5′OH group. Now, the only
chance of ligation is between the vector and the
foreign DNA resulting in a recombinant DNA molecule
as the insert DNA has the 5′PO4 group. Thus, self- 156
4. Gene Transformation/Transgenics/ Introduction of
rDNA into host Cell
• The ultimate aim of the development of recombinant DNA
molecules or vectors is the multiplication of rDNA by
cloning in a suitable host cell. Which mean that after the
construction of the recombinant DNA molecule, it has to
be introduced into a suitable host cell so that the DNA will
copy (multiply). There are several methods available for
this, which depend on several factors including the type
of vector used and the host cell.
• i.e. Gene cloning means making multiple copies of a
gene. There are several ways in which this can be done.
The principal methods are divided into two main
categories; these are:
• in vivo cloning: the gene is introduced into a cell and is
copied as the cell divides
• in vitro cloning: this does not take place in living cells but
the DNA is copied many times over using the polymerase
chain reaction (PCR). this process mimics the natural
semiconservative replication of DNA in a machine called a PCR
machine. 157
Host cells = the factories of cloning
• The hosts are the living systems or cells in which the
carrier of recombinant DNA molecule or vector can
be propagated. There are different types of host
cells-prokaryotic (bacteria) and eukaryotic (fungi,
animals and plants). Microorganisms are preferred as
host cells, since they multiply faster compared to
cells of higher organisms (plants or animals).
• The bacterium, E. coli
• was the first organism used in the DNA technology
experiments and continues to be the host of choice
by many workers.
• is the most extensively studied and widely used
organism in rDNA experiments.
• is the most commonly used prokaryotic host cell,
with a wide variety of different strains available for
particular applications.
158
Advantage of using E. coli as a
cloning vector
• is very simple, easy to handle, grows rapidly and is
able to accept and maintain a range of vectors.
• the doubling time of E. coli under ideal growth
conditions is 20 minutes. As the cell undergoes
multiplication, the rDNA within the cell also
undergoes multiplication independent of its
genome. Within a short time the number of cells
and the quantity of the rDNA will be very high.
• if the recombinant plasmids have appropriate
promoter signals for expression, a large quantity of
protein can also be purified from these systems.
• there are a large number of genetically defined
strains, which can be used freely for genetic
engineering experiment and expression of
recombinant proteins because they are disarmed
and so are non-infective and harmless. 159
• Other bacteria may be used as hosts for
gene cloning experiments, with examples
including species of Bacillus, Pseudomonas,
and Streptomyces. There are, however,
certain drawbacks with most of these.
Often there are fewer suitable vectors
available for use in such cells than is the
case for E. coli, and getting recombinant
DNA into the cell can cause problems.
• The major drawback however, is that E.coli
(or even other prokaryotic organisms)
cannot perform post-translational
modifications.

160
Eukaryotes as host cell
• The yeast Saccharomyces cerevisiae is one of the most
commonly used eukaryotic microbes in genetic
engineering. It has been used for centuries in the
production of bread and beer and has been studied
extensively. The organism is amenable to classical genetic
analysis, and a range of mutant cell types is available. In
terms of genome complexity, S. cerevisiae has about 3.5
times more DNA than E. coli. The complete genome
sequence is now known. Other fungi that may be used for
gene cloning experiments include Aspergillus nidulans and
Neurospora crassa.
• Despite the practical difficulties to work with and high cost
factor, Plant and animal cells (such as mouse cells)are may
also be used as hosts for gene manipulation experiments.
The advantage is that certain complex proteins which
cannot be synthesized by bacteria can be produced by
mammalian cells e.g. tissue plasminogen activator. This is
mainly because the mammalian cells possess the
machinery to modify the protein to the active form (post-
translational modifications). 161
• Unicellular algae such as Chlamydomonas reinhardtii
have all the advantages of microorganisms plus the
structural and functional organization of plant cells,
and their use in genetic manipulation will increase as
they become more widely studied. Other plant (and
animal) cells are usually grown as cell cultures,
which are much easier to manipulate than cells in a
whole organism. Mammalian cell lines in particular
are very important sources of cells from gene
manipulation procedures.
• Higher eukaryotes present a different set of problems
to the genetic engineer, many of which require
specialized solutions. Often the aim of a gene
manipulation experiment in a higher plant or animal
is to alter the genetic makeup of the organism by
creating a transgenic, rather than to isolate a gene
for further analysis or to produce large amounts of a
particular protein.
162
• After the construction of the recombinant DNA molecule, it has to be
introduced into a suitable host cell so that the DNA will multiply.
There are several methods available for this, which depend on
several factors including the type of vector used and the host cell.
• The following are common methods used for introducing the rDNA
molecule into the host cell:
a) Transformation
• Is the process of introducing DNA (usually plasmid DNA) into cells.
• i.e. the uptake of plasmid DNA by host cells is referred to as transformation.
• is the most commonly adopted method for introducing a recombinant
DNA into a host cell. During the process of transformation, cells take
up foreign DNA from their environment. But many cells including E.
coli, yeast, and mammalian cells are not able to take up the DNA
from their environment naturally. Certain chemical treatments can
enhance the ability of cells to take up the foreign DNA or make the
cell competent for transformation. This enhanced competency for
transformation was found out by Mandel and Higa in 1970. They
observed that E. coli cells become more competent to take up foreign
DNA when these cells are incubated in cold calcium chloride solution.
This mechanism is still followed for the transformation of E. coli cells
as a part of gene cloning.
• Transformation in bacteria was first demonstrated in 1928 by Frederick Griffith, in
his famous ‘transforming principle’ experiment that paved the way for 163
the
discoveries that eventually showed that genes were made of DNA.
• To effect transformation of E. coli, the cells need
to be made
competent. This is achieved by soaking the
cells in an ice-cold solution of calcium chloride,
which induces competence in a way that is still
not fully understood. Transformation of
competent cells is carried out by mixing the
plasmid DNA with the cells, incubating on ice for
20--30 min, and then giving a brief heat shock
(2 min at 42◦C
is often used), which appears to enable the DNA
to enter the cells. The transformed cells are
usually incubated in a nutrient broth at 37◦C for
60--90 min to enable the plasmids to become
established and permit phenotypic expression of
their traits. The cells can then be
plated out onto selective media for propagation
of cells harbouring the plasmid.
164
Transformation (the uptake of DNA by bacterial
cells)
• Most species of bacteria, including E. coli, take up only
limited amounts of DNA under normal circumstances.
In order to transform these species efficiently, the
bacteria have to undergo some form of physical and/or
chemical treatment that enhances their ability to take
up DNA. Cells that have undergone this treatment are
said to be competent, cells that are treated to increase
their ability to take up foreign DNA. This can be done
through various methods, such as chemical treatment
with calcium chloride or electroporation.

165
b) Transfection
• Is the process by which introduction of
purified phage or virus DNA into cells.
• i.e. transfection refers to the uptake of phage
DNA.
• is another method used for the
transformation of cultured cells. In this
method DNA (recombinant vector) is mixed
with charged substances such as calcium
phosphate, cationic liposomes, or DEAE
dextran and overlayered on the host cells.
This ultimately results in the uptake of the
external DNA by these host cells.

166
Introduction of phage DNA into bacterial
cells
• There are two different methods by which a
recombinant DNA molecule constructed with
a phage vector can be introduced into a
bacterial cell: transfection and in vitro
packaging.
• Transfection= is equivalent to transformation,
the only difference being that phage DNA rather
than a plasmid is involved. Just as with a plasmid,
the purified phage DNA, or recombinant phage
molecule, is mixed with competent E. coli cells and
DNA uptake induced by heat shock. Transfection is
the standard method for introducing the double
stranded replicative form of an M13 cloning vector
into E. coli.
167
In vitro packaging of λ cloning vectors
• Transfection with λ DNA molecules is not a very efficient
process when compared with the infection of a culture of
cells with mature λ phage particles. It would therefore be
useful if recombinant λ molecules could be packaged into
their λ head-and-tail structures in the test tube.
• This may sound difficult but is actually relatively easy to
achieve. Packaging requires a number of different
proteins coded by the λ genome, but these can be
prepared at a high concentration from cells infected with
defective λ phage strains. Two different systems are in
use. With the single strain system, the defective λ phage
carries a mutation in the cos sites, so that these are not
recognized by the endonuclease that normally cleaves
the λ catenanes during phage replication. This means
that the defective phage cannot replicate, though it does
direct synthesis of all the proteins needed for packaging.
The proteins accumulate in the bacterium and can be
purified from cultures of E. coli infected with the mutated
λ. The protein preparation is then used for in vitro
packaging of recombinant λ molecules. 168
• With the second system two defective λ strains are
needed. Both of these strains carry a mutation in a
gene for one of the components of the phage protein
coat: with one strain the mutation is in gene D, and
with the second strain it is in gene E. Neither strain is
able to complete an infection cycle in E. coli because
in the absence of the product of the mutated gene the
complete capsid structure cannot be made. Instead
the products of all the other coat protein genes
accumulate.
• An in vitro packaging mix can therefore be prepared
by combining lysates of two cultures of cells, one
infected with the λ D - strain, the other infected with
the E - strain. The mixture now contains all the
necessary components for in vitro packaging.
• With both systems, formation of phage particles is
achieved simply by mixing the packaging proteins with
λ DNA-assembly of the particles occurs automatically
in the test tube. The packaged λ DNA is then 169
170
• Most of the problems associated with
getting DNA into nonbacterial cells have
involved plant cells. Animal cells are
relatively flimsy and can be transformed
readily. However, plant cells pose the
problem of a rigid cell wall, which is a
barrier to DNA uptake. This can be
alleviated by the production of
protoplasts, in which the cell wall is
removed enzymatically. The protoplasts
can then be transformed using a technique
such as electroporation,
microinjection, and biolistic method.

171
c) Electroporation
• is another efficient method for introducing rDNA into a
host cell. During the process of electroporation an
electric current is used to create transient microscopic
pores in the cell membrane of the host cell. Through
these temporary openings foreign DNA enters the cell.
This method is especially suitable for cells such as
yeast, mammalian cells, and plant protoplasts.
d) Microinjection
• is a specialized technique by which the DNA fragment or
gene can be directly injected into the nucleus of plant
and animal cells. This method can be carried out without
the use of any specialized vector. The procedure
involves the direct injection of the DNA into the nucleus
of the host cell with the help of a glass microinjection
tube or syringe. The instrumentation for microinjection
consists of a microscope of high magnification, attached
with fine needles, syringes, and other facilities to carry
out the delivery of DNA into the selected cells 172
automatically.
e) The biolistic method
• was developed for introducing foreign DNA into plant cells
with the help of a gene or particle gun. During the
biolistic method, microscopic particles of gold or
tungsten coated with the DNA of interest is
bombarded into the cells at a high velocity. The
bombardment of the particle is carried out with the help of
a mechanical device called a particle gun.
f) Agrobacterium mediated
• Normally, the method of introducing foreign genes into the
plant cell is done using Agrobacterium tumifaciens. But this
bacterium is host specific and may not infect and transfer
the gene in certain cases, particularly in the case of
monocots. Biolistic methods can be adopted in such cases.

• In the case of bacteriophage vectors no special methods

or treatments are required for inserting the DNA into the
genome because the phage infection and the transfer of
DNA into the genome is natural. 173
174
5. Selection and Screening of
recombinants (clones containing gene of
interest)
• There are two terms that require definition
before we proceed:
 Selection
• is where some sort of pressure (e.g. the presence of
an antibiotic) is applied during the growth of host
cells containing recombinant DNA. The cells with the
desired characteristics are therefore selected by
their ability to survive.
• e.g. antibiotic or β-galactosidase status
• It can be Positive selection and Negative
selection
• Screening
• is a procedure by which a population of viable cells
is subjected to some sort of analysis that enables
the desired sequences to be identified. 175
• The introduction of recombinant DNA into a suitable
host cell is followed by the selection of those cells,
which contain the recombinant vectors. During the
process of genetic transformation only a very low
percentage of the total cell population takes or
receives the recombinant DNA. Therefore, it is very
important to have an efficient selection and
screening methods to select the transformed cells
or those cells that have taken the foreign DNA.
There are various selection methods or
screening techniques that are based on the
expression or non-expression of some of the
traits present in the vector or along with the
cloned gene. Some of these traits are the
resistance to certain antibiotics. If the antibiotic-
resistance gene is present along with the cloned
gene, it is very easy to select the recombinant
transformants directly on a medium supplemented
with the respective antibiotic. 176
Note:
• During rDNA process, in addition to
the desired recombinant molecule the
ligation mixture may contain, any of
the following:
• Host cells + Unligated vector molecules
• Host cells + Unligated DNA fragments
• Host cells + Vector molecules that have re-
circularized without new DNA being inserted
(“self-ligated” vector)
• Host cells + Recombinant DNA molecules that
carry the wrong inserted DNA fragment.
• Host cells + Recombinant DNA molecules that
carry the desired DNA fragment.
• Host cells without any plasmid
177
178
• In most of the cases there are three stages of
selection.
 First, one is the selection of non-transformed cells
• i.e., the cells that have not taken a vector from cells that
have taken a vector. i.e. selection of cells with and without
vector.
• Common method is antibiotics resistance test
 Second, one is the selection of transformed cells
(i.e., the cells that have taken a vector).
• i.e. selection of cells containing vector without insert and
cells containing vector with insert).
• Common method is Blue and White colony screening.
• using β-galactosidase/X-gal system.
 The third one is to identify the transformed cells
that have the recombinant plasmid. This method is
considered as Screening step
• i.e. cells that have the recombinant plasmid with target
insert or cells that have the recombinant plasmid without
target insert).
• Common methods are Colony hybridization or Southern
179
blotting techniques
• The host cells contain a plasmid and can be
identified by the positive selection method. One
of the simplest genetic selection methods involves the
use of antibiotics to select for the presence of vector
molecules.
• i.e. The most common markers used for positive selection
are the antibiotic-resistance genes present in the cloning
vectors.
• For example, the plasmid pBR322 contains genes for
ampicillin resistance (Apr) and tetracycline
resistance (Tcr). Thus, the presence of the plasmid in
cells can be detected by plating potential transformants
on an agar medium that contains either (or both) of
these antibiotics. Only cells that have taken up the
plasmid will be resistant, and these cells will therefore
grow in the presence of the antibiotic.
• i.e. Antibiotic resistance marker genes provide a simple and
reliable way to select for the presence of vectors in cells.
• Similarly, the ampicillin-resistance gene is present in
pUC19 and other similar types of vectors. 180
• A positive-selection method is very
important when the DNA is introduced
through a plasmid by transformation. Only
a very small percentage of the cells picks
up the plasmids and becomes antibiotic
resistant. So, a strong positive-selection
system can eliminate all the
untransformed cells and allow the
transformed cells to grow. By plating the
transformation products directly on
antibiotic plates, all untransformed
bacteria die and only those containing the
plasmid with the antibiotic-resistance
marker gene can grow to form colonies.

181
• By this selection method we will be able to get the
transformed cells with the plasmids. But it is not
possible to identify the transformed cells or colonies
having recombinant plasmids from the transformed
cells or colonies with parent plasmid without any
insert DNA fragment (non-recombinant vector).
• A negative-selection method can be adopted for
identifying the presence of recombinant vector
among the transformed cells. In order to identify
those plasmids carrying a foreign DNA fragment,
the multiple cloning site in the vector is engineered
in such a way that the insertion of a foreign DNA
strand disrupts the expression of a selectable
marker gene, a phenomenon known as insertional
inactivation. Two types of selectable markers are
used for negative selection. One is the insertional
inactivation of antibiotic resistance and the second
is the insertional inactivation of an enzymatic 182
activity.
• While the insertional inactivation of antibiotic-resistance works, it
requires a lot of manipulation picking the positively selected bacteria
and replating on negative selection media, etc. In addition to the tedium
of picking colonies, vectors such as pBR322 also suffer from a paucity of
convenient restriction sites at which to insert the foreign DNA
fragments. These limitations promote the development of a set of host-
vector systems in which it is possible to positively select for bacteria
carrying a plasmid and simultaneously select for insertional inactivation
of enzymatic activity. This system is based on the beta-galactosidose
gene (lac Z gene) of the E. coli lac operon. This method is based on the
insertional inactivation of lac Z gene. The lac Z gene is a component of
the lac operon and it produces an enzyme ꞵ-galactosidase. This enzyme
can cleave a colorless, synthetic substrate, X-gal into a blue-colored
product. If the β-galactosidase gene (lac Z gene) is disrupted and
inactivated by inserting the foreign DNA fragment into it, X-gal will not
be cleaved and the development of the blue color will be prevented.
Thus, the selection medium is provided with the X-gal and the host cells
that carry normal vectors (without insert DNA) will produce blue-colored
colonies, and those cells which carry the recombinant plasmids will
develop white-colored colonies. Here, the positive selection (expression
of antibiotic resistance) and the negative selection (inactivation of an
enzyme activity—ꞵ-galactosidase) can be combined together and
carried out in a single experiment. In addition to X-gal and antibiotics as
the means of selection additives, the media also require a compound
called IPTG as an inducer for the
expression of lac Z gene. 183
• In order to use antibiotic-resistance as a negative
as well as a positive-selection system, the plasmid
vector must carry two different antibiotic-
resistance genes. An example of such a vector is
pBR322. pBR322 carries both an ampicillin and a
tetracycline resistance gene. The phenotype of the
bacteria containing the intact
plasmid is Ampr Tetr (ampicillin resistant and
tetracycline resistant). Insertion of foreign DNA
into the PstI site located in the Ampr gene results
in an Amps Tetr (ampicillin susceptible and
tetracycline resistant) phenotype. Conversely,
insertion of foreign DNA into the EcoRI, Hind-III, or
SalI sites located in the Tetr gene results in an
Ampr Tets phenotype. In order to reveal these
phenotypes, transformants are first plated on the
positive selection media (Tet or Amp,
respectively). Positively selected colonies are then
re-streaked on the positive selection media 184
Selection stage 1
A. Selection using marker gene (Antibiotic
resistance test)= Positive selection
• Antibiotic resistance test (i.e. identifying the
clones containing plasmid vector and may or may
not contain DNA fragment from other source and
can survive (grow) on the media containing
antibiotics. This is also called positive selection
method.
• Mechanism of bacterial
selection that contain
a selectable marker
(antibiotic resistance
gene)

185
• B. Selection using Blue and white colony
selection= Negative Selection (This technique
also called the use of chromogenic substrates for
selection of recombinant clones)
• Clones showing a positive test in Antibiotic
resistance test are identifying whether they
contain DNA fragment from other source or not on
the media containing x-gal. This is also called
negative selection method.
• If the clone do not contain DNA fragment in its
plasmid vector, the media turns to blue. Those
clones are not selected for further screening.
• If the clone contain DNA fragment in its plasmid
vector from other source, the colony become white
because the Lac Z gene is interrupted and the β-
galactosidase is not produced. Those clones are
selected for further screening.
186
Blue and white colony screening
• Blue colonies
• represent antibiotic-resistant bacteria
that contain plasmid vector and express
a functional alpha fragment from an
intact LacZ alpha coding sequence.
• White colonies
• represent antibiotic-resistant bacteria
that contain plasmid insert and do not
produce LacZ alpha fragment
Insertion of a DNA fragment interrupts
the open reading frame (ORF) of lacZ’
gene, resulting in non-functional gene
product
This that known
phenomenon can not digest its
as insertional
substrate x-gal.
inactivation 187
• The use of chromogenic substrates in
genetic screening methods has been an
important aspect of the development of the
technology. The most popular system uses the
compound X-gal (5-bromo-4-chloro- 3-indolyl--
D-galactopyranoside), which is a colorless
substrate for β-galactosidase. The enzyme is
normally synthesized by E. coli cells when
lactose becomes available. However, induction
can also occur if a lactose analogue such as
IPTG (iso-propyl-thiogalactoside) is used. This
has the advantage of being an inducer without
being a substrate for β-galactosidase. On
cleavage of X-gal a blue-coloured product is
formed; thus, the expression of the lacZ (β-
galactosidase) gene can be detected easily.
188
• The previous slide indicates that, the X-gal system
can be used as a screen for cloned sequences. If a
DNA fragment is cloned into a functional β-
galactosidase gene (e.g. into the EcoRI site of
Charon 16A), any recombinants will be
genotypically lacZ- and will therefore not produce
β-galactosidase in the presence of IPTG and X-gal.
Plaques containing such phage will therefore
remain colourless. Non-recombinant phage will
retain a functional lacZ gene and, therefore, give
rise to blue plaques. This approach can also be
used with the -complementation system; in this
case the insert DNA inactivates the lacZ region in
vectors such as the M13 phage and pUC plasmid
series. Thus, complementation will not occur in
recombinants, which will be phenotypically Lac-
and will therefore give rise to colourless plaques
or colonies
189
• The X-gal detection system can be used where a
functional β- galactosidase gene is present in the
host/vector system. This can occur in two ways. First, an
intact β-galactosidase gene (lacZ) may be present in the
vector, as is the case for the insertion vector Charon
16A. Host cells that are Lac- are used for propagation of
the phage, so that the Lac+ phenotype will only arise
when the vector is present. A second approach is to
employ the α-complementation system, in which only
part of the lacZ gene (encoding a peptide called the α-
peptide) is carried by the vector. The remaining part of
the gene sequence is carried by the host cell. The region
coding for the smaller, vector-encoded α-peptide is
designated lacZ. Host cells are therefore designated
lacZ-. Blue colonies or plaques will only be produced
when the host and vector fragments complement each
other to produce functional β-galactosidase.
• The α-peptide of β-galactosidase can be used in a vector
to enable a more sophisticated form of X-gal blue/white
identification to be achieved by α-complementation.
190
What is the concept of α-complementation
• α-complementation system, in which only part of
the lacZ gene (encoding a peptide called the α-
peptide) is carried by the vector. The remaining
part of the gene sequence is carried by the host
cell. The region coding for the smaller, vector-
encoded α-peptide is designated lacZ. Host cells
are therefore designated lacZ -. Blue colonies or
plaques will only be produced when the host and
vector fragments complement each other to
produce functional β galactosidase.
• i.e. The portion of the lacZ gene encoding the first
146 amino acids (the α-fragment) are on the plasmid.
The remainder of the lacZ gene is found on the
chromosome of the host. If the α-fragment of the lacZ
gene on the plasmid is intact (that is, you have a non-
recombinant plasmid), these two fragments of the
lacZ gene (one on the plasmid and the other on the
chromosome) complement each other and will
produce a functional β-galactosidase enzyme. 191
192
 lacZ encode enzyme β-galactosidase

lac promoter X-gal (substrate of the

enzyme)

IPTG
IsoPropyl-β-D-Thiogalactoside (as an inducer
Blue product
for the expression of lac Z gene)

 During the experiment,

IPTG is added to the
selective growth medium.
Insertion of the target DNA
fragment into the Multiple
cloning site (MCS) will
prevent the formation of
the blue colony, and white 193
 Vector with no insert: blue
transformants
Recombinant plasmid (colony
containing inserted DNA): white
transformants
vector no insert

Recombinant plasmid
(contain insert)

Thus, the color of the colony allows quick determination of whether

a recombinant or intact plasmid is present in the cell. 194
195
196
The rationale behind
insertional
inactivation of the
lacZ′ gene carried by
pUC8.
a) The bacterial and
plasmid genes
complement each other
to produce a functional
β-galactosidase
molecule.
b) Recombinants are
screened by plating
onto agar containing
197 X-
• Insertional inactivation approach of selecting
recombinants by Antibiotic resistance test
• Beside for the first method used for selection of
host cells whether they contain vector or not
Antibiotic resistance status can be also used to
identify a vector carrying a cloned insert using
Insertional inactivation approach. i.e. the
presence of cloned DNA fragments can be
detected if the insert interrupts the coding
sequence of a gene. This approach is known as
insertional inactivation and can be used with
any suitable genetic system. If the insert is cloned
into the coding sequence of an antibiotic
resistance gene, the gene will be inactivated and
the cell will be sensitive to the antibiotic. This is a
very powerful method of selecting cells for further
analysis.
198
• Antibiotic resistance can be used as an insertional
inactivation system if DNA fragments are cloned into
a restriction site within an anitbiotic-resistance gene.
For example, cloning DNA into the PstI site of pBR322
(which lies within the Apr gene) interrupts the coding
sequence of the gene and renders it non-functional.
Thus, cells that harbour a recombinant plasmid will be
ApsTcr. This can be used to identify recombinants as
follows: if transformants are plated first onto a
tetracycline-containing medium, all cells that contain
the plasmid will survive and form colonies. If a replica
of the plate is then taken and grown on ampicillin-
containing medium, the recombinants (ApsTcr) will not
grow, but any non-recombinant transformants (AprTcr)
will. Thus, recombinants are identified by their
absence from the replica plate and can be picked
from the original plate and used for further analysis.

199
 The clone contain the DNA fragment in its
plasmid vector (showing white colony in X-
gal media) needs further screening to
identify whether the fragment found in the
plasmid is the target gene (gene of
interest) or not.
Screening stage 2
To identify the clone containing the DNA fragment
whether it is target gene or not
Method 1-by Restriction fragment analysis by
Southern blotting to screen the recombinants
(clones containing gene of interest) using probes
(complementary to the target gene)
Method 2- by colony hybridization method using
probes (complementary to the target gene)
200
Method I: Restriction fragment analysis by Southern blotting to screen the
recombinants (clones containing gene of interest)

201
The DNA is separated on a gel, then transferred (“blotted”) onto a
solid support medium such as nitrocellulose paper or a nylon
membrane. It is then incubated with a radioactive single-strand
copy of the gene of interest, which hybridizes to the blot at the
location(s) where there is a fragment with a complementary
sequence. The positions of radioactive bands on the blot identify
the fragments of interest.

202
• As described in the above figure probes are
used to determine the location of specific DNA
fragments in a gel by a Southern blot.
• i.e. in the procedure the fractionated DNA
fragments are washed out of the gel and trapped
onto a nitrocellulose membrane, which is
incubated with radioactively labeled DNA (or RNA)
probes. The location of the hybridized fragments is
determined autoradiographically. During the
blotting procedure, capillary action draws the
buffer upward into the paper towels. As the buffer
moves through the electrophoretic gel, it dissolves
the DNA fragments and transfers them to the
203
surface of the adjacent membrane.
Method II: Colony hybridization for detection of
clones contains desired gene

204
Using a DNA probe
to identify a cloned
gene in a
population of
bacteria

205
Screening by Colony Nucleic Acid
Hybridization

206
Identification/ screening of
recombinant phages
Eg.1. Insertional inactivation of a
lacZ′ gene carried by the phage
vector
• All M13 cloning vectors, as well as several e
vectors, carry a copy of the lacZ′ gene.
Insertion of new DNA into this gene
inactivates ꞵ-galactosidase synthesis, just
as with the plasmid vector pUC8.
Recombinants are distinguished by plating
cells onto X-gal agar: plaques comprising
normal phages are blue; recombinant
plaques are clear.
207
e.g.2. Insertional inactivation of the λ cI gene
and resulted in change of plaque morphology
can also be used as a screening method for certain
vectors such as gt10, which contain the cI gene. This
gene encodes the cI repressor, which is responsible
for the formation of lysogens. Plaques derived from
cI+ vectors will be slightly turbid because of the
survival of some cells that have become lysogens. If
the cI gene is inactivated by cloning a fragment into a
restriction site within the gene, the plaques are clear
and can be distinguished from the turbid non-
recombinants. This system can also be used as a
selection method.
• i.e. Insertional inactivation of cI gene causes a change in
plaque morphology. Normal plaques appear “turbid”,
whereas recombinants with a disrupted cI gene are
“clear”. The difference is readily apparent to the
experienced eye.

208
Immunological screening for expressed
genes
• An alternative to screening with nucleic acid
probes is to identify the protein product of a
cloned gene by immunological screening.
The technique requires that the protein is
expressed in recombinants, and is most often
used for screening cDNA expression libraries
that have been constructed in vectors such as
gt11. Instead of a nucleic acid probe, a
specific antibody is used.

209
Strategies for the selection of
recombinant phage

210
211
Chapter 2
Enzymes in Genetic Engineering
and their Mechanism of Action
 Restriction Nucleases (Restriction
Enzymes)
• Exonucleases
• Endonucleases
 Enzymes In Modification
• Polynucleotide Phosphorylase, Dnase
• Phosphatases and Methylases
• Ligases, Polynucleotide Kinase, Rnase
212
Overview of this chapter
• Once pure samples of DNA have been prepared, the
next step in a gene cloning experiment is construction
of the recombinant DNA molecule. To produce this
recombinant molecule, the vector, as well as the DNA
to be cloned, must be cut at specific points and then
joined together in a controlled manner. Cutting and
joining are two examples of DNA manipulative
techniques, a wide variety of which have been
developed over the past few years. As well as being
cut and joined, DNA molecules can be shortened,
lengthened, copied into RNA or into new DNA
molecules, and modified by the addition or removal of
specific chemical groups. These manipulations, all of
which can be carried out in the test tube, provide the
foundation not only for gene cloning, but also for
studies of DNA biochemistry, gene structure, and the
control of gene expression. 213
DNA manipulative enzymes
• can be grouped into four broad classes,
depending on the type of reaction that
they catalyze:
1. Polymerases
• make copies of molecules.
2. Modifying enzymes
• remove or add chemical groups.
3. Nucleases
• are enzymes that cut, shorten, or
degrade nucleic acid molecules.
4. Ligases
• join nucleic acid molecules together.
214
DNA Polymerases
• are enzymes that synthesize a new strand of DNA
complementary to an existing DNA or RNA template. Most
polymerases can function only if the template possesses a
double-stranded region that acts as a primer for initiation of
polymerization.
• E.g. DNA polymerase I, which is usually prepared from E. coli.
This enzyme attaches to a short single-stranded region (or nick) in
a mainly double-stranded DNA molecule, and then synthesizes a
completely new strand, degrading the existing strand as it
proceeds. DNA polymerase I is therefore an example of an enzyme
with a dual activity DNA polymerization and DNA degradation.
• The polymerase and nuclease activities of DNA polymerase I are
controlled by different parts of the enzyme molecule. The nuclease
activity is contained in the first 323 amino acids of the
polypeptide, so removal of this segment leaves a modified enzyme
that retains the polymerase function but is unable to degrade DNA.
This modified enzyme, called the Klenow fragment, can still
synthesize a complementary DNA strand on a single-stranded
template, but as it has no nuclease activity it cannot continue the
synthesis once the nick is filled in. Several other enzymes-natural
polymerases and modified versions-have similar properties to the
Klenow fragment. The major application of these polymerases215is in
DNA sequencing.
• The Taq DNA polymerase used in the polymerase
chain reaction (PCR) is the DNA polymerase I enzyme
of the bacterium Thermus aquaticus. This organism
lives in hot springs, and many of its enzymes,
including the Taq DNA polymerase, are thermostable,
meaning that they are resistant to denaturation by
heat treatment. This is the special feature of Taq DNA
polymerase that makes it suitable for PCR, because if
it was not thermostable it would be inactivated when
the temperature of the reaction is raised to 94°C to
denature the DNA.
• The final type of DNA polymerase that is important in
genetic engineering is reverse transcriptase, an
enzyme involved in the replication of several kinds of
virus. Reverse transcriptase is unique in that it uses
as a template not DNA but RNA. The ability of this
enzyme to synthesize a DNA strand complementary
to an RNA template is central to the technique called
complementary DNA (cDNA) cloning. 216
The reactions catalyzed by DNA polymerases. (a) The basic reaction: a new
DNA strand is synthesized in the 5’ to 3’ direction. (b) DNA polymerase I,
which initially fills in nicks but then continues to synthesize a new strand,
217
degrading the existing one as it proceeds. (c) The Klenow fragment, which
DNA modifying enzymes
• There are numerous enzymes that modify
DNA molecules by addition or removal of
specific chemical groups. The most important
are as follows:
1.Alkaline phosphatase, which removes the
phosphate group present at the 5′ terminus of a
DNA molecule.
2.Polynucleotide kinase, which has the reverse
effect to alkaline phosphatase, adding phosphate
groups onto free 5′ termini.
3.Terminal deoxynucleotidyl transferase, which
adds one or more deoxyribonucleotides onto the
3′ terminus of a DNA molecule.

218
The reactions catalyzed by DNA modifying enzymes. (a) Alkaline
phosphatase, which removes 5′-phosphate groups. (b) Polynucleotide
kinase, which attaches 5′-phosphate groups. (c) Terminal deoxynucleotidyl
219
transferase, which attaches deoxyribonucleotides to the 3′ termini of
Nucleases
• Nucleases degrade DNA molecules by
breaking the phosphodiester bonds
that link one nucleotide to the next in
a DNA strand.
• There are two different kinds of
nuclease:
• Exonucleases
• remove nucleotides one at a time from the
end of a DNA molecule.
• Endonucleases
• are able to break internal phosphodiester
bonds within a DNA molecule.
220
The reactions catalyzed by the
two different kinds of
nuclease.
(a) An exonuclease, which
removes
nucleotides from the end of a
DNA
molecule.
(b) An endonuclease, which
breaks internal phosphodiester
bonds.

The reactions catalyzed by

different types of
exonuclease.
(a) Bal31, which removes
nucleotides from both
strands of a double-
stranded molecule.
(b)(b) Exonuclease III, which
221
Endonucleases
• also called Restriction endonuclease or
Restriction enzymes
Types of Restriction Endonucleases
• At present more than 2500 different
restriction endonucleases have been
identified that are isolated from a number of
species of bacteria.
• More than 300 restriction enzymes are now
available for use in the laboratory.
• These enzymes belong to three different
classes of restriction endonucleases that can
be distinguished from each other on the basis
of their structure, cofactors required and
features of their restriction and cleavage site.
222
Type I
• Type I restriction enzymes have a complex structure with three different subunits
(endonuclease, methyl transferase and recognition).
• These enzymes cut DNA at a random site, far (~1 kb) from the recognition
sequence. Their restriction sites are also complex and discontinuous with spacers.
• They may have biological significance but are of very little practical value since
the site where the DNA is going to be digested cannot be predicted.
Type II
• Type II restriction endonucleases cleave DNA at very precise and defined positions
close to or within the recognition sequence.
• These enzymes require magnesium (Mg2+) ions as cofactors for their activity.
• They are smaller in size, in comparison to type I and type III enzymes and mostly
bind to DNA as homodimers.
• Their recognition site is mostly symmetric, showing two fold symmetry.
• Most of the type II enzymes cleave with in the symmetric recognition sequence,
e.g. HhaI, HindIII and NotI, but there are other classes also that cleave outside
their recognition sequence.
• A few other type II restriction enzymes cleave on both sides of the recognition
sequence releasing a small fragment.
Type III
• This class of enzymes are large and complex proteins that cleave outside the
recognition sequence.
• The cleavage is non-specific and ~30 nucleotides away from the recognition
sequence. 223
224
Characteristic features of different classes of restriction enzymes

225
Nomenclature of Restriction
endonuclease
• Three-letter acronym for each enzyme
derived from the source organism
• First letter from genus
• Next two letters represent species
• Additional letter or number represent the
strain or serotypes
• The Roman numeral represents the order of
discovery
For example. the enzyme
• HindII was isolated from Haemophilus
influenzae serotype d.
• EcoRI was isolated from Escherichia coli
serotype R. 226
227
• Restriction endonuclease are Bacterial enzymes that cut
DNA at specific sites.

• How bacteria possesses these enzymes? To protect the bacteria

itself from Bacteriophage

• What kinds of bonds are broken when restriction enzymes cut

DNA?
A. Phosphodiester bonds
B. Hydrogen bonds
C. Both
228
Why Restriction Enzymes Works in
Bacteria?
• Phage (or viruses) invade all types of cells.
Bacteria are one favorite target.
• Defense mechanisms have been
developed by bacteria to defend
themselves from these invasions.
• Bacteria have evolved a class of enzymes
that destroy foreign DNA (eg. Virus DNA).
▫ protect bacteria from bacteriophages (Viruses).
• Infecting DNA is cleaved (restricted) by the
restriction enzyme(s) preventing it from
successfully replicating and parasitizing
the cell.

229
 Why the bacteria does not kill itself by its own
Restriction Enzymes? i.e. If everything gets
cleaved, how come the bacteria does not kill itself?

AnS: Due to the restriction enzyme modification

systems

• Usually, organisms that make restriction enzymes

also make a companion modification enzyme (DNA
methyl transferase-methylase) that protects their
own DNA from cleavage. i.e. Bacteria protect their
self DNA from restriction digestion by methylation
of its recognition site. Methylation is adding a
methyl group (CH3) to DNA.
• These enzymes recognize the same DNA sequence
as the restriction enzyme they accompany, but
instead of cleaving the sequence, they disguise it
by methylating one of the bases in each DNA
strand. 230
the restriction enzyme modification systems
adding a methyl group (CH3) to DNA= by DNA methyl transferase-methylase

231
 - Restriction endonucleases - recognize
pallindromic sequences
- Palindrome = a word, verse, or sentence
that reads the same forwards or backwards.

 Complete the following palindrome sequence and name the

restriction endonuclease that recognizes this.
5' --------G ? A ? T C------ 3'
3' ---------C ? T ? A G------ 5'
232
Examples of Restriction Enzymes
Enzyme Organism source Recognized
Sequence

EcoRI Escherichia coli 5' GAATTC 3’

3' CTTAAG 5’

TaqI Thermus aquaticus 5' TCGA 3’

3' AGCT 5’
HindIII Haemophilus influenzae 5'AAGCTT 3’
3'TTCGAA 5’
BamHI Bacillus amyloliquefaciens 5' GGATCC 3’
3' CCTAGG 5’
AluI Arthrobacter luteus 5' AGCT 3’
3' TCGA 5’
233
The frequency of restriction
endonuclease cut sites
i.e. The frequency of recognition sequences of
restriction endonuclease in a DNA molecule
• The number of recognition sequences for a
particular restriction endonuclease in a DNA
molecule of known length can be calculated
mathematically.
• A tetranucleotide sequence (e.g., GATC)
should occur once every 44 = 256
nucleotides, and a hexanucleotide (e.g.,
GGATCC) once every 46 = 4096 nucleotides.
• Note: Restriction sites are generally not evenly spaced out along a
DNA molecule.

234
How to estimate the frequency of
the RE in a DNA molecule or
genome?
Random occurrence of cut site of endonuclease
can be calculated by the general formula =
n
1/4
n= number of bases
e.g. The random occurrence of the hexameric (6)
sequence:
is 1/46 =1/4096

235
236
• Restriction enzymes cleave DNA in the form of:
1. Sticky (cohesive) ends (i.e. when restriction
endonucleases cleave both strands of DNA at the alternate
phosphodiester bonds and leaving unpaired bases on the
ends),
• Sticky (cohesive) ends are an end of a double-
stranded DNA molecule where there is a single-
stranded extension. i.e. DNA strands are cut at
cleavage is staggered, so that the resulting DNA
fragments have short single-stranded overhangs at
each end.
• Sticky ends increase the efficiency of ligation. This
is because compatible sticky ends can base pair with one
another by hydrogen bonding, forming a relatively stable
structure for the enzyme to work on. If the phosphodiester
bonds are not synthesized fairly quickly then the sticky ends
fall apart again. These transient, base-paired structures do,
however, increase the efficiency of ligation by increasing the
length of time the ends are in contact with one another.

237
2. Blunt (flush) ends (i.e.
when restriction endonucleases cleave
both strands of DNA at the opposing
phosphodiester bonds and leaving no
unpaired bases on the ends)

238
239
Recognition sequences and cut sites of various endonucleases

Restriction cleavage with Sma-I

resulting in blunt ends.

Blunt ends

Sticky ends

240
The restriction site sequence of EcoRI is a palindrome of a hexamer and it cuts between the bases G
and A on both the strands resulting in sticky ends or cohesive ends

241
242
• If restriction endonucleases cleave both
strands of DNA at the opposing
phosphodiester bonds and leaving no
unpaired bases on the ends (blunt
ends), how can we prepare rDNA?
• There are three methods that can be used to
put the correct sticky ends onto the blunt
DNA fragments.
• Using
linkers
Adaptors
homopolymer tailing
243
Generating a new RE cut site at a
blunt end
• Using linkers
• Linkers are
• short blunt ended synthetic dsDNA
containing a RE cut site
• used to place sticky ends on to a blunt-
ended DNA molecule.
• Experimental step:
i. Blunt ended DNA + linker
ii. Digest with appropriate RE
iii. Ligate to vector

244
 245
Linkers and their use: (a) the structure of a typical linker; (b) the attachment of
246
• Drawback of linkers= Consider what would happen
if the blunt-ended molecule contained one or more
BamHI recognition sequences. If this was the case,
the restriction step needed to cleave the linkers and
produce the sticky ends would also cleave the blunt-
ended molecule. The resulting fragments will have
the correct sticky ends, but that is no consolation if
the gene contained in the blunt-ended fragment has
now been broken into pieces.

A possible problem with the use of linkers. Compare this situation with
the desired result of BamHI restriction 247
Adaptors
• The second method of attaching sticky ends
to a blunt-ended molecule is designed to
avoid this problem. Adaptors, like linkers,
are short synthetic oligonucleotides. But
unlike linkers, an adaptor is synthesized so
that it already has one sticky end.
• Drawback = the sticky ends of
individual adaptor molecules could
base pair with each other to form
dimers.

248
Adaptors and the potential problem with their use. (a) A typical adaptor.
(b) Two adaptors could ligate to one another to produce a molecule
similar to a linker, so that (c) after ligation of adaptors a blunt-ended
molecule is still blunt-ended and the restriction step is still needed. 249
• Obviously, each end of a double-stranded
molecule consists of one 5′-P terminus and one 3′-
OH terminus and Ligation takes place between the
5′-P and 3′-OH ends. That is why adapter make
dimers. However, after the 5′-P terminus is
modified: one of the adapter end lacks the
phosphate group, and it become a 5′-OH terminus.
Hence, DNA ligase is unable to form a
phosphodiester bridge between 5′-OH and 3′-OH
ends and no dimer formation. Therefore, after the
adapter is modified it can be ligated to a blunt-
ended DNA molecule but not to themselves. After
the adaptors have been attached, the abnormal
(modified) 5′-OH terminus is converted to the
natural 5′-P form by treatment with the enzyme
polynucleotide kinase, producing a sticky-ended
fragment that can be inserted into an appropriate
vector.
250
The use of adaptors: (a) the actual structure of an adaptor, showing the
modified 5′-OH terminus; 251
Producing sticky ends by homopolymer tailing
• The technique of homopolymer tailing offers a radically
different approach to the production of sticky ends on a
blunt-ended DNA molecule. A homopolymer is simply a
polymer in which all the subunits are the same.
• Tailing involves using the enzyme terminal deoxynucleotidyl
transferas to
add a series of nucleotides onto the 3′-OH termini of a
double-stranded DNA molecule. If this reaction is carried out
in the presence of just one deoxyribonucleotide, a
homopolymer tail is produced. Of course, to be able to ligate
together two tailed molecules, the homopolymers must be
complementary. Frequently polydeoxycytosine (poly(dC))
tails are attached to the vector and poly(dG) to the DNA to
be cloned. Base pairing between the two occurs when the
DNA molecules are mixed.
• In practice, the poly(dG) and poly(dC) tails are not usually
exactly the same length, and the base-paired recombinant
molecules that result have nicks as well as discontinuities.
Repair is therefore a two-step process, using Klenow
polymerase to fill in the nicks followed by DNA ligase 252to
synthesize the final phosphodiester bonds. This repair
Homopolymer tailing:
(a) synthesis of a
homopolymer tail;
(b)construction of a
recombinant DNA
molecule from a
tailed vector plus
tailed insert DNA;
(c) repair of the
recombinant DNA
molecule.
253
Blunt end ligation with a DNA topoisomerase
Blunt end ligation with a DNA topoisomerase. (a) Cleavage of the vector
with the topoisomerase leaves blunt ends with 5′-OH and 3′-P termini. (b)
The molecule to be cloned must therefore be treated with alkaline
phosphatase to convert its 5′-P ends into 5′-OH termini. (c) The
topoisomerase ligates the 3′-P and 5′-OH ends, creating a double-
stranded molecule with two discontinuities, which are repaired by cellular
enzymes after introduction into the host bacteria.

DNA
topoisomerases
have both
nuclease
(double-stranded
breakages) and
ligase activities.

254
DNA ligase
• The study of DNA replication and repair processes led
to the discovery of the DNA-joining enzyme called DNA
ligase. DNA ligases catalyze formation of a
phosphodiester bond between the 5′-phosphate of a
nucleotide on one fragment of DNA and the 3′-hydroxyl
of another. This joining of linear DNA fragments
together with covalent bonds is called ligation. Unlike
the type II restriction endonucleases, DNA ligase
requires ATP as a cofactor. Because it can join two
pieces of DNA, DNA ligase became a key enzyme in
genetic engineering. If restriction-digested fragments of
DNA are placed together under appropriate conditions,
the DNA fragments from two sources can anneal to
form recombinant molecules by hydrogen bonding
between the complementary base pairs of the sticky
ends. However, the two strands are not covalently
bonded by phosphodiester bonds. DNA ligase is
required to seal the gaps, covalently bonding the two
strands. 255
• The DNA ligase most widely used in the lab
is derived from the bacteriophage T4. T4
DNA ligase will also ligate fragments with
blunt ends, but the reaction is less efficient
and higher concentrations of the enzyme
are usually required in vitro. To increase the
efficiency of the reaction, researchers often
use the enzyme terminal deoxynucleotidyl
transferase to modify the blunt ends. For
example, if a single-stranded poly(dA) tail is
added to DNA fragments from one source,
and a single stranded poly(dT) tail is added
to DNA from another source, the
complementary tails can hydrogen bond.
Recombinant DNA molecules can then be
created by ligation. 256
 Generally, Ligase Enzymes- are
used to put together DNA (i.e. catalyze the
formation of a phosphodiester bond between
adjacent 3’-OH and 5’-P group in DNA).
Action of DNA ligase
Two polynucleotide chains, one with a free 3-OH group
and one with a free 5-phosphate group, are joined by
DNA ligase, which forms a phosphodiester bond.

257
Ligation: the final step in construction of a recombinant DNA molecule.
Q. In a DNA ligation, why is it
important to inactivate the restriction
enzymes before adding the DNA
ligase?
Ans. If this is not done, the enzymes
will continue to recut the DNA every
time the DNA ligase seals the gap.

258
259
Chapter 3
Nucleic acid hybridization and
amplification
• Methods of Nucleic Acid Detection
• Polymerase Chain Reaction (PCR) and
its Applications
• Variations in PCR and their
Applications
• Probe and Target Sequences
• Methods of Nucleic Acid Hybridization

260
Methods of Nucleic Acid Detection
• Nucleic acid detection methods are essential tools in
molecular biology, genetics, and diagnostics. Here are
some of the primary methods used:
1. Normal Polymerase Chain Reaction (PCR)
• Description: Amplifies specific DNA sequences, making
millions of copies from a small sample.
• Applications: Disease diagnosis, genetic testing, forensic
analysis.
2. Quantitative PCR (qPCR)
• Description: A variation of PCR that quantifies DNA in real-
time.
3. Reverse Transcription PCR (RT-PCR)
• Description: Converts RNA into DNA (via reverse
transcription) before amplification.
• Applications: Studying RNA expression, detecting RNA
viruses.
4. Southern Blotting
• Description: Detects specific DNA sequences in a sample.
261
5. Northern Blotting
• Description: A technique for detecting specific RNA sequences
in a sample.
• Applications: Studying gene expression, RNA processing.
• Applications: Measuring gene expression levels, viral
load in infections.
6. In Situ Hybridization (ISH)
• Description: Uses labeled RNA or DNA probes to detect
specific nucleic acids in tissue sections.
• Applications: Localizing gene expression in tissues,
studying developmental biology.
7. CRISPR-based Detection
• Description: Utilizes CRISPR technology to identify specific
nucleic acid sequences.
• Applications: Rapid diagnostics, pathogen detection.
8. Microarray Analysis
 Description: Allows simultaneous detection of thousands
of nucleic acid sequences.
 Applications: Genomic studies, expression profiling.
262

9. etc.
 Polymerase chain reaction (PCR) and its
Applications
• PCR
• It was originally invented by Kary Mullis in 1985.
• is a fundamental technique in molecular biology that
allows for the amplification of specific DNA sequences.
• is a very quick, easy, automated method used to
amplifying (make copies) of a specific segment of DNA
• i.e. PCR is a process that ‘amplifies’ or ‘copies’ a piece of
DNA repeatedly until there is an amount which is great
enough to observe visually. i.e. it can generate millions of
copies of a target DNA fragment from a tiny sample.
 The DNA fragment to be amplified should not be
greater than about 3 kb in length and ideally less than
1 kb. Fragments up to 10 kb can be amplified by
standard PCR techniques, but the longer the fragment
the less efficient the amplification and the more difficult
it is to obtain consistent results. Amplification of very
long fragments-up to 40 kb-is possible, but requires
special methods. 263
 Materials/components required for
PCR
1. Template DNA (The original DNA which is
being amplified)
2. DNA primers that “bracket” the desired
sequence to be cloned
3. Heat-resistant DNA polymerase (Taq
Polymerase)
4. DNA nucleotides (dNTPs)
5. Thermocycler (PCR machine)

264
• The Taq DNA polymerase is a temperature-tolerant
enzyme isolated from thermus aquaticus, a bacterium
found in hot springs. It catalyzes the synthesis of DNA.
This enzyme is stable and active at near-boiling
temperatures. Therefore, it is well suited for carrying
out PCR reactions.

265
266
Steps of PCR
 PCR amplification of DNA occurs by repeated cycles (25-
30) of three temperature dependent steps
1.Denaturation
• The dsDNA template is heated (denatured) to break the
hydrogen bonds between the two polynucleotide
strands by heating the sample to 94-950C.
2. Annealing:
• Oligonucleotide primers are annealed to the ssDNA
template when lowering the temperature at about 50-
600C.
3. Primer extension (Polymerization or DNA
synthesis):
• requires thermo-stable DNA polymerase that withstand
repeated exposure to high temperatures (i.e. Taq
polymerase)
• The enzyme Taq polymerase is added to extends each
primer in the 5' to 3' direction at a temperature of
72°C. Source of Taq polymerase= from a bacterium found in hot
springs
• The rate of extension is about 50-100 nts/sec. Thus, 267the
A typical temperature profile for a PCR

268
 100 Melting 30x
94 oC
Temperature Extension
Annealing 72 oC
Primers
50
50 oC

0
T i m e

PCR Reaction

269
270
271
Diagram representation of the exponential amplification of selected DNA
sequences during PCR reaction at every cycle.
272
DNA Between The Primers Doubles With Each
Thermal Cycle
Number
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles
273
274
Total number of DNA copies during PCR is determined by

the formula =2 ,
n
n= number of cycles

275
Exercise

A human DNA fragment of 3 kb is to be

amplified by PCR. The total genome size
is 3 x 109 bp.
a) Prior to amplification, what fraction of
the total DNA does the target sequence
constitute?
b) What fraction does it constitute after 10
cycles of PCR?
c) After 20 cycles of PCR?
d) After 30 cycles of PCR?

276
Designing the oligonucleotide primers for a PCR
• The primers are the key to the success or failure of a PCR
experiment. If the primers are designed correctly the experiment
results in amplification of a single DNA fragment, corresponding
to the target region of the template molecule. If the primers are
incorrectly designed the experiment will fail, possibly because no
amplification occurs, or possibly because the wrong fragment, or
more than one fragment, is amplified. Clearly a great deal of
thought must be put into the design of the primers.
The results of PCRs with well designed
and poorly designed primers.
• Lane 1 shows a single amplified
fragment of the expected size, the
result of a well designed experiment.
• In lane 2 there is no amplification
product, suggesting that one or both
of the primers were unable to
hybridize to the template DNA.
• Lanes 3 and 4 show, respectively, an
amplification product of the wrong
size, and a mixture of products (the
correct product plus two wrong ones);
277
both results are due to hybridization
• Working out appropriate sequences for the primers is not a
problem: they must correspond with the sequences flanking
the target region on the template molecule. Each primer must,
of course, be complementary (not identical) to its template
strand in order for hybridization to occur, and the 3′ ends of
the hybridized primers should point toward one another.
• Parameters should be considered during designing of
primers for PCR are:
1) length of the primers
2) annealing temperature
• The first important issue to address is the length of the
primers.
• If the primers are too short they might hybridize to non-target sites
and give undesired amplification products. To illustrate this point,
imagine that total human DNA is used in a PCR experiment with a pair
of primers eight nucleotides in length (in PCR jargon, these are called
“8-mers”). The likely result is that a number of different fragments will
be amplified. This is because attachment sites for these primers are
expected to occur, on average, once every 4 8 = 65,536 bp, giving
approximately 49,000 possible sites in the 3,200,000 kb of nucleotide
sequence that makes up the human genome. This means that it
would be very unlikely that a pair of 8-mer primers would give a
278
single, specific amplification product with human DNA.
• What if the 17-mer primers shown in Figure are used? The
expected frequency of a 17-mer sequence is once every 4 17 =
17,179,869,184 bp. This figure is over five times greater than
the length of the human genome, so a 17-mer primer would
be expected to have just one hybridization site in total human
DNA. A pair of 17-mer primers should therefore give a single,
specific amplification product

279
The lengths of the primers are critical for the specificity
• Why not simply make the primers as long as possible?
• The length of the primer influences the rate at which it
hybridizes to the template DNA, long primers hybridizing at a
slower rate. The efficiency of the PCR, measured by the number
of amplified molecules produced during the experiment, is
therefore reduced if the primers are too long, as complete
hybridization to the template molecules cannot occur in the time
allowed during the reaction cycle. In practice, primers longer
than 30-mer are rarely used.
• In addition to the size of the primer, the annealing temperature
is also important parameter because, again, this can affect the
specificity of the reaction. DNA–DNA hybridization is a
temperature-dependent phenomenon. If the temperature is too
high no hybridization takes place; instead the primers and
templates remain dissociated. However, if the temperature is
too low, mismatched hybrids ones in which not all the correct
base pairs have formed are stable. If this occurs the earlier
calculations regarding the appropriate lengths for the primers
become irrelevant, as these calculations assumed that only
perfect primer–template hybrids are able to form. If mismatches
are tolerated, the number of potential hybridization sites for
each primer is greatly increased, and amplification is more likely
to occur at non-target sites in the template molecule. 280
• The ideal annealing temperature must be low
enough to enable hybridization between primer
and template, but high enough to prevent
mismatched hybrids from forming This temperature
can be estimated by determining the melting
temperature or Tm of the primer–template
hybrid. The Tm is the temperature at which the
correctly base-paired hybrid dissociates (“melts”).
A temperature 1–2°C below this should be low
enough to allow the correct primer–template hybrid
to form, but too high for a hybrid with a single
mismatch to be stable. The Tm can be determined
experimentally but is more usually calculated from
In which
the [G + C]
simple is the number of G and C nucleotides in
formula.
the primer sequence, and
[A + T] is the number of A and T nucleotides.
The annealing temperature for a PCR experiment is therefore
determined by calculating the Tm for each primer and using a
temperature of 1–2°C below this figure. Note that this means the
two primers should be designed so that they have identical Tms. If
this is not the case, the appropriate annealing temperature for281 one
This indicates that temperature has an important effect on the hybridization
of the primers to the template DNA 282
e.g. Calculate the Tm of the following
primer.

283
After the PCR: studying PCR products
• PCR is often the starting point for a longer
series of experiments in which the
amplification product is studied in various
ways in order to gain information about the
DNA molecule that acted as the original
template.
• Although a wide range of procedures have
been devised for studying PCR products,
three techniques are particularly important:
1. Gel electrophoresis of PCR products
2. Cloning of PCR products
3. Sequencing of PCR products

284
Applications of PCR
• Applicable in almost all areas of molecular biology, genetics, and in
clinical areas.
• Including
• Medical diagnostics: PCR is used to diagnose a variety of infectious diseases
(detection of bacterial & viral infection).
• What is the advantage of PCR over conventional biochemical tests to identify the
bacteria?
• Detect genetic mutations associated with certain diseases. i.e. the genetic
mutations responsible for certain genetic diseases and cancers can be detected very
early and the possibility of causing the disease can be predicted and treated or
precautionary measures can even be taken before the disease manifests. Early
detection of genetic disease is even possible in embryonic conditions or even in sex
cells-sperm and egg.
• Detection of microorganisms in food samples, water, and in the environment
with the help of species-specific primers.
• For DNA fingerprinting purpose (forensics): PCR is used to identify individuals
from blood, hair, or other biological samples ()
• To analyze DNA evidence in criminal cases (criminology or forensics): The DNA
of the suspects and the DNA sample recovered from the crime scene can be
analyzed by PCR techniques with the help of a set of identical random primers or
specific primers.
• Gene cloning: PCR is used to amplify DNA fragments for cloning into plasmids or
other vectors. This is a critical step in many gene expression and protein production
experiments.
• Gene sequencing: PCR is often used to amplify DNA fragments for sequencing.
This allows researchers to determine the order of nucleotides in a DNA molecule.
285
• etc.
Application of PCR in Medical diagnostics
e.g. Diagnosis of AIDS by PCR method

• The T-lymphocytes of a suspected AIDS patient are

isolated and disrupted to release DNA. The so
obtained DNA is amplified by PCR and to this DNA
probes are added. If the HIV DNA is present, it
hybridizes with the complementary sequence of the
labeled DNA probe which can be detected by its
radioactivity.
• Therefore, the advantage of DNA probe for
diagnosis is that it can detect the virus when there
are no detectable antibodies in the circulation.
• For example, diagnosis of HIV in the new born (baby).i.e. because
the new born baby cannot produce antibody until 18 month.
Hence, if we want to check HIV in the new born using antibody
test we cannot say a new born is HIV +ve. Because the antibody
found in the new born is comes from the mother so we detect the
HIV comes from mother rather than the new born.
286
Variations in PCR and their
Applications
• Over the years, several variations of PCR have been
developed to enhance its capabilities and address
specific research requirements. i.e. Being a versatile
technique, PCR is modified as per the specific
demands of the situation. Some of the variants of
PCR are listed

1. Real-time quantitative PCR (qPCR)

2. Reverse transcription PCR (RT-PCR)
3. Nested PCR
4. Inverse PCR
5. Anchored PCR
6. Asymmetric PCR
7. etc 287
1. Real-time quantitative PCR (qPCR)
• Quantitative PCR, also known as real-time PCR,
• is a powerful technique used to amplify and simultaneously quantify a
targeted DNA molecule. It allows researchers to measure the amount of
DNA in real-time during the PCR process, making it a valuable tool in
molecular biology and diagnostics.

Unlike traditional PCR, where the amplification is assessed at the end of the
process, qPCR measures the fluorescence emitted during the reaction,
providing real-time quantification (Real-Time Monitoring).
 qPCR utilizes fluorescent dyes or probes that emit a signal during
amplification, enabling real-time monitoring of the amplification process.
i.e. qPCR typically utilizes fluorescent dyes or probes that bind to the DNA.
Commonly used dyes include:
 SYBR Green: Binds to double-stranded DNA and emits fluorescence.
 TaqMan Probes: Specific probes that emit fluorescence when
cleaved during the amplification process.
To quantify the initial amount of DNA, researchers often create standard
curves using known concentrations of target DNA. i.e To determine the
concentration of the starting template DNA the fluorescent signal
throughout the reaction is compared to a standard curve of amplified DNA
of a known starting concentration. The cycle in which the unknown DNA is
detected compared to the standard curve can be used to determine the
amount of starting material in your sample. 288
Applications od qPCR
• Gene Expression Analysis: Quantifying mRNA
levels to study gene expression under various
conditions. i.e qPCR helps them understand which
genes are being actively expressed in a cell.
• Pathogen Detection: Diagnosing infectious
diseases by quantifying pathogen DNA/RNA
(microbial load quantification, and genotyping). E.g.
qPCR is used in viral load determination, such as
monitoring HIV or COVID-19 infections.
• Genetic Variation Analysis: Identifying single
nucleotide polymorphisms (SNPs) and mutations.
• Copy Number Variation: Assessing variations in
gene copy numbers in different samples.
• qPCR has applications in gene expression analysis,
microbial load quantification, and genotyping.

289
Advantages
• Sensitivity: Can detect low amounts of
nucleic acids.
• Specificity: High specificity due to the use
of primers and probes.
• Speed: Provides results within a few hours.
• The development of real time PCR has
resulted in a great decrease in the time needed
for detection of DNA from infectious
microorganisms.
• Classical methods often required 3 or 4 days to
isolate the microorganism and another week to
confirm its identity always assuming the
pathogen can be cultured.
• Quantitative: Allows for precise
quantification of nucleic acids.
290
291
2. Reverse-transcription PCR (RT-PCR)
• Another PCR procedure called reverse-
transcription PCR uses viral RNA or a cell's
mRNA as the template. The enzyme,
reverse transcriptase, makes DNA from the
RNA template, and the DNA is then
amplified. Because RNA is a very unstable
molecule, so it must first be converted into cDNA
• is used to amplify RNA molecules (templates) by
(complementary DNA) using an enzyme called
converting them into complementary DNA
reverse transcriptase.
(cDNA) using the enzyme reverse transcriptase.
• It is commonly used to study gene expression
levels, detect RNA viruses (such as COVID-19),
and analyze RNA-based biomarkers.
• It can be useful in determining low levels of gene
expression by analyzing the PCR product of cDNA 292

prepared from the mRNA transcript.

293
294
295
How conventional PCR is different from RT–PCR?
• RT–PCR is a variation of PCR, or polymerase chain
reaction. The two techniques use the same process
except that RT–PCR has an added step of reverse
transcription of RNA to DNA, or RT, to allow for
amplification. This means PCR is used for pathogens,
such as viruses and bacteria, that already contain
DNA for amplification, while RT–PCR is used for those
containing RNA that needs to be transcribed to DNA
for amplification. Both techniques can be performed
in ‘real time’, which means results are visible almost
immediately, while when used ‘conventionally’,
results are only visible at the end of the reaction.

296
How does RT–PCR work with the COVID-19 virus?
• A sample is collected from the parts of the body where
the COVID-19 virus gathers, such as a person’s nose or
throat. The sample is treated with several chemical
solutions that remove substances such as proteins and
fats and that extract only the RNA present in the sample.
This extracted RNA is a mix of the person’s own genetic
material and, if present, the virus’ RNA.
• The RNA is reverse transcribed to DNA using a specific
enzyme. Scientists then add additional short fragments of
DNA (probes) that are complementary to specific parts of
the transcribed viral DNA. If the virus is present in a
sample, these fragments attach themselves to target
sections of the viral DNA. Some of the added genetic
fragments are used for building DNA strands during
amplification, while the others are used for building the
DNA and adding marker labels to the strands, which are
then used to detect the virus.

297
• The mixture is then placed in an RT–PCR machine. The machine
cycles through temperatures that heat and cool the mixture to
trigger specific chemical reactions that create new, identical
copies of the target sections of viral DNA. The cycle is repeated
over and over to continue copying the target sections of viral
DNA. Each cycle doubles the previous number: two copies
become four, four copies become eight, and so on. A standard
real time RT–PCR set-up usually goes through 35 cycles, which
means that, by the end of the process, around 35 billion new
copies of the sections of viral DNA are created from each strand
of the virus present in the sample.
• As new copies of the viral DNA sections are built, the marker
labels attach to the DNA strands and then release a fluorescent
dye, which is measured by the machine’s computer and
presented in real time on the screen. The computer tracks the
amount of fluorescence in the sample after each cycle. When a
certain level of fluorescence is surpassed, this confirms that the
virus is present. Scientists also monitor how many cycles it
takes to reach this level in order to estimate the severity of the
infection: the fewer the cycles, the more severe the viral
infection is.
• i.e. During the amplification process, the probe anneals to a specific target
sequence located between the forward and reverse primers. During 298 the
extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase
299
3. Nested PCR
• is a two-step PCR procedure that is used to
increase the specificity of the reaction. In the
first step, a pair of primers is used to amplify a
large region of DNA. In the second step, a new
pair of primers, nested within the first pair, is
used to amplify a smaller, more specific region of
DNA. Nested PCR is used to detect rare DNA
sequences or to amplify DNA from samples that
contain contaminants.
• i.e it involves two rounds of amplification. In the first
round, outer primers amplify the target region, and
in the second round, inner primers amplify a specific
region within the product of the first round. Nested
PCR enhances specificity and sensitivity and is
commonly used for detecting low-abundance
targets, such as in forensic DNA analysis or
pathogen detection.
300
Nested PCR is a useful way of overcoming some of
the problems associated with a large number of PCR
cycles, which can lead to error prone synthesis.

301
A diagram illustrating the method of nested PCR. 302
4. Inverse PCR
• Often a stretch of DNA sequence is known, but the desired target
sequence lies outside this region. This causes problems with primer
design, as there may be no way of determining a suitable primer
sequence for the unknown region. Inverse PCR (IPCR) involves
isolating a restriction fragment that contains the known sequence
plus
flanking sequences. By circularizing the fragment, and then cutting
inside the known sequence, the fragment is effectively inverted.
Primers can then be synthesized using the known sequence data
and
used to amplify the fragment, which will contain the flanking
regions.
Primers that face away from each other (with respect to direction
of product synthesis) in the original known sequence are required
so that, on circularization, they are in the correct orientation. The
technique can also be used with sets of primers for nested PCR.
Deciphering the result usually requires DNA sequencing to
determine the
areas if interest. An illustration of IPCR is shown in the following
figure

303
304
• Generally, Inverse PCR involves designing
primers that anneal to the known region and
amplify outward into the unknown regions.
• Inverse PCR is useful for genome walking,
identifying integration sites of transposons, or
determining the surrounding sequences of known
DNA fragments.
305
5. Multiplex PCR
• Multiplex PCR enables the simultaneous
amplification of multiple target sequences in a single
reaction. It uses multiple primer sets, each specific
to a different target sequence.
• Multiplex PCR is used commonly in disease or
pathogen identification. Scientists can
simultaneously detect several different pathogens in
one specimen saving both time and effort.
• i.e. Multiplex PCR is valuable for detecting multiple
pathogens in a single sample, genotyping, and mutation
analysis.
• i.e. Using Multiplex PCR more than one target sequence
can be amplified by using multiple primer pairs in a
reaction mixture.
• As an extension to the practical use of PCR, this technique
has the potential to produce considerable savings in time
and effort within the laboratory without compromising on
the utility of the experiment.
306
307
• 6. Hot-start PCR: Hot-start PCR involves temporarily
inactivating the DNA polymerase before the reaction
starts. This prevents nonspecific amplification and
primer-dimer formation during the initial setup. Hot-
start PCR improves specificity and can be used for
amplifying challenging templates or reducing
background noise.
• 7. High-fidelity PCR: High-fidelity PCR utilizes DNA
polymerases with proofreading activity that have a
lower error rate during DNA synthesis. This variation
is employed when high accuracy and fidelity in DNA
amplification are crucial, such as in cloning,
sequencing, or site-directed mutagenesis.
• etc

308
Probe and Target Sequences
• In the context of nucleic acid hybridization, the terms "probe" and
"target sequence" refer to the two key components involved in the
hybridization process.
Target Sequence:
• The target sequence is the nucleic acid (DNA or RNA) molecule that
the researcher wants to detect or analyze.
• The target sequence can be present in a complex mixture, such as
genomic DNA, cDNA, or total RNA.
• The target sequence must be complementary to the probe sequence
for successful hybridization to occur.
• The target sequence can be obtained from various sources, such as
biological samples, cell cultures, or experimental materials.
Probe Sequence:
• The probe is a single-stranded nucleic acid (DNA or RNA) molecule
with a known sequence.
• The probe is designed to be complementary to the target sequence
that the researcher wants to detect or analyze.
• Probes can be labeled with various reporter molecules, such as
radioactive isotopes, fluorescent dyes, or enzymes, to facilitate the
detection of the hybridization event.
• Probes can be generated through various methods, including309 PCR
Probes
• are nucleotide sequences that are complementary to some
part of the target gene.
• generally refers to a nucleic acid (usually DNA) that has the
same or a similar sequence to that of a specific gene or
DNA sequence of interest, such that the denatured probe
and target DNA can hybridize when they are renatured
together.
• are usually a single-stranded DNA of defined sequence that
is distinctively labeled, either with a radioactive isotope
(such as 32P) or some other easily detectable tag. The
nucleotide sequence of the probe is designed to be
complementary to the sought-for or target DNA fragment.
• types of probe:
1. Oligonucleotide probes, which are synthesized
chemically and end-labeled;
2. DNA probes, which are cloned DNAs and may either
be end-labeled or internally labeled during in vitro
replication; and 310

3. RNA probes (riboprobes), which are internally

DNA probes
• a synthetic single stranded DNA molecule that can recognize and specifically bind to a
target DNA by complementary base paring in a mixture of molecules.
• a radioactive single-strand DNA molecule which is complementary to the target
gene.
• used to screen the recombinants (clones containing gene of interest)
• i.e. searching for a specific cloned DNA sequence in a library is called library
screening. One of the key elements required to identify a gene during library
screening is the probe.
• i.e. a radioactive single-strand copy of the gene of interest, which hybridizes to
the blot at the location(s) where there is a fragment with a complementary
sequence. The positions of radioactive bands on the blot identify the fragments
of interest.
• A DNA probe is a small, fluorescently or radioactively labeled DNA molecule that is
used to locate similar or complementary sequences among a long stretch of DNA
molecule or bacterial colonies such as genomic or cDNA libraries or in a genome.
Such DNA probes are used in hybridization experiments such as Southern
hybridization to detect certain specific sequences, which are complementary to the
probes. Since the probe is labeled with a fluorescent dye or radioactive isotopes of
phosphorous, its binding to specific sequences can be detected. DNA probes labeled
with radioactive isotopes or fluorescent dyes can be used for the screening of
transformed colonies having the correct recombinant plasmid by Southern
hybridization.
• i.e. is important to determining the location of specific DNA fragments in a gel by
a Southern blot.
• RNA probes and oligonucleotide probes are generally single-stranded. DNA
may be labeled as a double-stranded or single-stranded molecule, but it is only
useful as a probe when single stranded and therefore must be denatured before 311
use.
Probes can be also categorized into two major types:
1) Heterologous probes
• A heterologous probe is a probe that is similar to, but
not exactly the same as, the nucleic acid sequence of
interest. If the gene being sought is known to have a
similar nucleotide sequence to a second gene that has
already been cloned, then it is possible to use this
known sequence as a probe. For example, a mouse
probe could be used to search a human genomic library.
• i.e. A piece of DNA from the corresponding gene in a related
organism can also be used as a probe in screening a library
for a particular gene. Such probes are termed
heterologous probes because they are not derived from
the homologous (same) organism.
2) Homologous probes
• A homologous probe is a probe that is exactly complementary to
the nucleic acid sequence of interest. Homologous probes can be
designed and constructed in a number of different ways.
Examples include degenerate probes, expressed sequence tag
(EST) based probes, and cDNA probes that are used to locate a
genomic clone. 312
Nucleic acid hybridization
• Any two single-stranded nucleic acid molecules will attempt to base
pair with one another under appropriate conditions. In most cases,
the hybrid structures thus formed will be very unstable since the total
number of H-bonds that formed is very low. However, if there is
significant sequence complementarity between the two strands,
stable hybrids will be formed. This phenomenon is termed as
nucleic acid hybridization.
• is a fundamental technique in molecular biology and genetics that
involves the formation of complementary base pairing between two
single-stranded nucleic acid molecules, typically DNA or RNA.
• Here are some of the common methods used for nucleic acid
hybridization:
• Southern Blotting:
• This technique is used to detect and analyze specific DNA sequences within a
complex mixture of DNA fragments.
• It involves the separation of DNA fragments by size using gel electrophoresis,
transfer of the fragments to a membrane (e.g., nitrocellulose or nylon), and
hybridization with a labeled DNA probe.
• Northern Blotting:
• This method is used to detect and analyze specific RNA molecules within a
complex mixture of RNA.
• It involves the separation of RNA molecules by size using gel electrophoresis,
transfer of the RNA to a membrane, and hybridization with a labeled DNA or
313 RNA
probe.
• In Situ Hybridization (ISH):
• This technique allows the localization and visualization of
specific nucleic acid sequences within cells or tissues.
• It involves the use of labeled DNA or RNA probes that
hybridize to their complementary sequences in fixed,
permeabilized cells or tissue sections.
• There are different types of ISH, such as fluorescence in situ
hybridization (FISH) and chromogenic in situ hybridization
(CISH).
• Microarray Hybridization:
• Microarrays are high-throughput platforms that allow the
simultaneous detection and quantification of thousands of
nucleic acid sequences.
• In microarray hybridization, labeled DNA or RNA samples are
hybridized to an array of immobilized, complementary DNA
or RNA sequences (probes) on a solid surface.

314
The process of nucleic acid hybridization
involves the following steps:
• Denaturation:
• The target nucleic acid is denatured, usually by
heat or chemical treatment, to separate the
double-stranded molecules into single strands.
• Hybridization:
• The labeled probe is added to the denatured
target sequence. The probe and target sequence
form complementary base pairs, creating a
stable hybrid duplex.
• Detection:
• The hybridized probe-target complex is detected
using the reporter molecule attached to the
probe, such as radioactivity, fluorescence, or
enzymatic activity. 315
Blotting techniques (Southern blotting;
Western blotting, Northern blotting)
• Blotting=refers to the process of immobilization of
nucleic acids or proteins on solid support (nitrocellulose
or nylon membranes).
• DNA, protein, or RNA can be transferred to a
nitrocellulose filter (by placing the filter on the gel and
allowing the buffer flow from the gel into the filter to
carry with DNA, protein or RNA. After DNA, protein, or
RNA is transferred to the filter, the filter can be incubated
with radioactively labeled DNA fragments known as
probes. The probes will hybridize with complementary
DNA, protein, or RNA sequences, if they are present. The
filter then is exposed to x-ray film and any bound probes
are detected on the film, in a process called an
autoradiogram. This procedure is known as Southern
blotting, Western blotting, and Northern blotting when
DNA, protein or RNA is transferred to a filter respectively.
Western blotting involves the transfer of protein blots and
their identification by using specific antibodies. 316
Southern Blotting
• This technique was originally devised by Edward Southern in
1975 to identify specific DNA fragments on an agarose gel
after the separation by electrophoresis.
• The technique involves following procedures
• The isolation of the genomic DNA with a suitable
procedure.
• The genomic DNA is digested with a suitable restriction
enzyme or with a mixture of different restriction enzymes
to cut the long DNA molecule into fragments.
• Restriction-digested genomic DNA is separated by
electrophoresis
• The DNA separated on the gel is then transferred to a
membrane-a nitrocellulose or nylon membrane-with a
process called blotting, which is driven by capillary
action.
• The DNA fragments transferred to the membrane are
immobilized to its surface by heat or UV mediated cross-
linking.
• Now the membrane is hybridized with a ‘labeled’ segment
317
• Southern hybridization allows the precise
localization of a given DNA sequence in a
genome, once a restriction map has
been constructed. If the probe is labeled with
a fluorescent chemical then the fluorescent
band can be visualized after illuminating the
nylon or nitrocellulose membrane with UV
light after the process of blotting.
• Since the DNA bands on the membrane will
have the same pattern as that present in the
gel, the position of a labeled band on the
membrane can be traced to the same
corresponding position in the gel.

318
319
Southern blotting procedures

320
Steps of Southern blotting

321
Diagram representation of the various steps in the technique of Southern
322
• There are a number of variations of Southern
hybridization or Southern blotting suited for specific
types of experiments. Some of them are dot blots,
colony hybridization, slot blots, etc., which are
comparatively easier.

• Similar to Southern hybridization there are altered

forms of molecular hybridization techniques
developed for RNA and proteins. There are the
techniques developed in which RNA is hybridized with
its complementary DNA sequences or a specific
protein such as antibody hybridized to another protein
such as its ligand or antigen. Since hybridization of
DNA with another DNA molecule is called Southern
hybridization or blotting, hybridization between RNA
and DNA is called Northern hybridization and
protein-protein hybridization such as antigen-antibody
binding or protein-ligand binding is called Western
hybridization. 323
Southern Blotting for diagnosis
application
• It is the technique developed for detection
of pathogens in the sample and genetic
disorders problems based on hybridization
principles.

• This technique can be accomplished by two

major strategies
• Colony hybridization
• to detect pathogens without amplification
and restriction digestion process
• Gel-electrophoresis based
• with or without amplification (PCR) process
324
Basic steps of Southern Blotting techniques for clinical
diagnosis application
• Colony/culture based • Gel based with or without PCR
without PCR technique technique
• Clinical sample • Clinical sample
• Culture to make colony • DNA isolation and restriction
digestion
• Cell lysis to release DNA
• Direct Gelelectrophoresis or
• DNA denaturation
DNA amplification with PCR
• Blotting (transferring of • DNA denaturation
DNA to the paper)
• Blotting (transferring of DNA
• Hybridization (the probe to the paper)
and the DNA from • Hybridization (the probe and
sample (colony)
the DNA from sample
• Wash to remove the • Wash to remove the unbound
unbound probes probes
• Detection • Detection
• Analysis In this case the probe may be specifically
• Analysis
In this case the probe may be designed for the detection of pathogenic
specifically designed for the bacteria or to detect genetic disorder
detection of pathogenic bacteria problems.

325
 Colony hybridization for detection Using a DNA probe to
of pathogenic bacteria identify pathogenic
bacteria in a population

Patient’s Probes designed

for specific
sample
pathogenic bacteria

326
327
• Example of Gel based Southern blotting technique for
diagnosis of genetic disorder (e.g. Cystic fibrosis) by
using mutated cystic fibrosis gene as a probe
In this technique
1. Human DNA is first digested with a restriction enzyme,
yielding thousands of fragments of various sizes.
2. The different fragments are then separated by gel
electrophoresis.
3. The fragments are put in a well at one end of a layer of
agarose gel.
4. Then an electrical current is passed through the gel. While
the charge is applied, the different-sized pieces of DNA
migrate through the gel at different rates. The fragments are
called RFLPs, for restriction fragment length polymorphisms.
5. DNA denaturation (separation of dsDNA into single stranded
form)
6. The separated fragments are transferred onto a filter by
southern blotting.
7. The fragments on the filter are then exposed to a ready 328
made radioactive probe or made from the cloned gene of
329
Note: the probe used for hybridization purpose in southern
blotting can be prepared in the form of solution or it can be in
the form of cloned gene in the host cells.

330
Fluorescent in situ hybridization (FISH)
• is a powerful technique used to detect and localize
specific DNA or RNA sequences in cells or tissues.
• relay on the complementary binding of a fluorescent
labeled nucleotide probe that can bind only to a
specific target sequence of DNA or RNA.
• can show gene deletions, duplications, amplifications,
and translocations by evaluating chromosomes.
• needs fluorescent microscope
• multiple target areas can be evaluated using multi-
color probes.
• Important for detection of
• Breast Cancer
• Leukemia
• Down syndrome (trisomy 21). Extra (third) copy of
chromosome 21
• Intracellular parasites such as viruses 331
General steps involved in FISH:
1.Sample Preparation:
 Collect and fix the cells or tissue samples using formaldehyde or alcohol.
 Embed the samples in paraffin or freeze them, depending on the type of
analysis.
2.Slide Preparation:
 Mount the samples onto glass slides.
 If using frozen sections, ensure they are thinly sliced.
3.Denaturation:
 Treat the samples with a denaturing solution (e.g., heat or alkaline solution) to
separate the DNA strands.
4.Probe Hybridization:
 Apply fluorescently labeled DNA or RNA probes that are complementary to the
target sequence.
 Incubate the slides under controlled conditions (temperature and time) to allow
hybridization.
5.Washing:
 Rinse the slides to remove unbound probes, typically using a series of washing
steps with decreasing stringency.
6.Imaging:
 Examine the slides using a fluorescence microscope.
 Capture images of the fluorescent signals to analyze the presence and location
of the target sequences.
7.Data Analysis:
332
 Interpret the results based on the fluorescence patterns and intensities
Summary on Basic steps of FISH
 Fixation of the cell or tissue using formaldehyde

 Preparation or designing of the appropriate probe

(DNA or RNA probe)

 Denaturation of the probe and the target DNA

 Hybridization of the probe and the target molecule

 Detection of the result with fluorescent microscope

333
334
Clinical application of FISH
• Prenatal testing (genetic counseling) for
chromosomal abnormalities
• Diagnosis of mutation underlying diseases like 335
336
Chapter 4

Construction of DNA library

 Construction of Genomic Library
 Construction of cDNA Library
 Screening and Preservation of DNA
Libraries

337
• What does it mean DNA library?

 It is collections of DNA fragments, typically

cloned into vectors, that represent the genetic
material of an organism or a specific tissue
 i.e. It is a collection of cloned DNA fragments.
 DNA library can be prepared into two different ways using two
different vectors, plasmid or lambda phage.

• Types of DNA Library

1. Genomic library &
2. cDNA library
338
1) Genomic DNA library
• The term genomic library is often used to describe a
set of clones representing the entire genome (a
collection of all the DNA sequences of a given species)
of an organism
• i.e. A genomic library contains DNA fragments that represent
the entire genome of an organism.
• genomic DNA library is a set of host cells that
collectively contain all of the DNA sequences from the
genome of another organism.
• Generally, a genomic library is a set of recombinant clones
that contains all of the DNA present in an individual
organism.
• An E. coli genomic library, for example, contains all the E. coli
genes, so any desired gene can be withdrawn from the library
and studied. Genomic libraries can be retained for many years,
and propagated so that copies can be sent from one research
group to another.
 Cloning DNA, by whatever method, gives rise to a
population of recombinant DNA molecules, often in
plasmid or phage vectors, maintained either in bacterial
339
• The first consideration in constructing a genomic
library is the number of clones required. This
depends on a variety of factors, the most obvious
one being the size of the genome. Thus, a small
genome such as that of E. coli will require fewer
clones than a more complex one such as the
human genome. The type of vector to be used
also has to be considered, which will determine
size of fragments that can be cloned. In practice,
library size can be calculated quite simply on the
basis of the probability of a particular sequence
being represented in the library.

340
341
E.g. In humans, the genome size is approximately 3 × 109 bp. With an
average insert size of 20 kb, the number of random fragments to ensure
with high probability (95–99%) that every sequence is represented is
approximately 106 clones for humans. The maths actually works out to 1.5
× 105 (i.e. (3 × 109 bp)/(2 × 104 bp)) but more clones are needed in
practice, since insertion is random. Bacteriophage λ or cosmid vectors are
typically used for genomic libraries. Since a larger insert size can be
accommodated by these vectors compared with plasmids, there is a
greater chance of cloning a gene sequence with both the coding sequence
342
and the regulatory elements in a single clone.
• When dealing with size of library, phage or
cosmid vectors are usually essential, as the
cloning capacity and efficiency of these
vectors is much greater than that of
plasmid vectors. Although cosmids, with the
potential to clone fragments of up to 47 kb,
would seem to be the better choice,
replacement vectors are often used. A wide
range of vectors may be used for cloning
genomic DNA,
although in practice the choice is
determined by the requirements of the
procedure and the size of
the fragments to be cloned for library
construction. This is because they are
easier to use than cosmid vectors, and this
343
Steps of genomic DNA library construction

1. Preparation of plasmid or phage vector

2. Extraction of total DNA from the organism

3. Cut DNA with RE

4. Insertion of a DNA fragment to an

appropriate vector (i.e. making rDNA)

5. Cloning in a bacterial cells

• This approach is called shotgun cloning because

the strategy has no way of targeting a particular
gene but instead seeks to clone all the genes of the
organism at one time. 344
345
346
Genomic Library

347
2) cDNA library
• is a set of host cells that collectively contain all the DNA
sequences produced by reverse transcriptase from the
mRNA obtained from cells of a particular type. Thus, a
cDNA library contains all the genes expressed in that cell
type, at the stage of differentiation when the mRNA was
isolated.
• Lack non-coding region of the genome (introns)
• The principle behind cDNA cloning is that an mRNA
population isolated from a specific tissue, cell type, or
developmental stage (e.g. embryo mRNA) should contain
mRNAs specific for any protein expressed in that cell type
or during that stage, along with “housekeeping” mRNAs
that encode essential proteins such as the ribosomal
proteins, and other mRNAs common to many cell types or
stages of development. Thus, if mRNA can be isolated, a
small subset of all the genes in a genome can be studied.
mRNA cannot be cloned directly, but a cDNA copy of the
mRNA can be cloned.
• Because a cDNA library is derived from mRNA, the library 348
Steps of cDNA library preparation
 isolation of mRNA from a specific tissue, cell
type, or developmental stage (e.g. embryo
mRNA)
 synthesis of ss cDNA from each mRNA (by
reverse transcriptase)
 conversion of ss cDNA to ds cDNA
 incorporation of cDNA into a vector
(plasmid or phage)
 ligation of the restriction cleaved ds cDNA
to plasmid or lambda phage (rDNA
formation)
 introducing recombinant vector into host
cells (e.g. E. coli for cloning) to produce a
library 349
cDNA library construction

350
Isolation of mRNA from the cell

351
352
353
Q. What are the two main differences
between a genomic and a cDNA library?

Ans. In a genomic library, at least in theory,

all the sequences from the genome are
equally represented. However, a cDNA library
only contains sequences for genes expressed
in the starting material. This means that
some genes may be highly represented while
others are completely absent from the library.
All of the sequences in the genome, including
promoters and terminators, introns and junk
DNA will be included in a genomic library
whereas a cDNA library will only contain
coding regions.
354
Screening and Preservation of DNA
Libraries
• Proper screening and preservation techniques of DNA
library are crucial for the long-term maintenance and
accessibility of DNA libraries, which are essential
resources for various applications in genomics, gene
discovery, and functional studies.
• Screening of DNA libraries involves the identification
and isolation of specific DNA fragments or clones of
interest from the larger library.
• i.e. Searching for a specific cloned DNA sequence in a library
is called library screening. One of the key elements required
to identify a gene during library screening is the probe.
• Probe = is a set of known nucleic acids whose
identity and genetic sequence is known. It is used to
hybridize to a target nucleic acid in order to identify it
and measure it. If the nucleic acid of interest is
present, the probe nucleic acid will hybridize with it.
355
Methods for screening DNA libraries include:
• Colony/Plaque Hybridization:
• Radioactive or fluorescently labeled DNA probes are used to detect and
identify colonies or plaques containing the target DNA sequence.
• i.e. particular recombinant DNA clones in a library can be
detected by colony or plaque hybridization with labeled
probes.
• PCR Screening:
• Polymerase chain reaction (PCR) is used to amplify and detect the
presence of specific DNA sequences within the library.
• Antibody Screening:
• In some cases a library is screened with a protein. For example, when a
cDNA library is being screened an antibody can be used to identify the
protein that is being expressed by the insert of the clone. In this case,
the library is said to be “incubated” with the antibody probe, not
hybridized.
• i.e Specific antibodies are used to detect and isolate clones expressing the
target protein.
• i.e. antibodies can be also used DNA library screening if an expression vector is
used.
• Southern blotting technique screening:
• Functional Screening:
• Clones are tested for a specific biological activity or function to identify
356
the desired DNA sequence.
Screening a genomic library by colony hybridization (or
plaque hybridization)
• A common method of screening plasmid-based genomic
libraries is to carry out a colony hybridization experiment. The
protocol is similar for phage-based libraries except that
bacteriophage plaques, not bacterial colonies, are screened.
In a typical experiment, host bacteria containing either a
plasmid based or bacteriophage-based library are plated out
on a petri dish and allowed to grow overnight to form colonies
(or in the case of phage libraries, plaques). A replica of the
bacterial colonies (or plaques) is then obtained by overlaying
the plate with a nitrocellulose disc. The disc is removed,
treated with alkali to dissociate bound DNA duplexes into
single-stranded DNA, dried, and placed in a sealed bag with
labeled probe. If the probe DNA is duplex DNA, it must be
denatured by heating at 70°C. The probe and target DNA
complementary sequences must be in a single stranded form
if they are to hybridize with one another. Any DNA sequences
complementary to probe DNA will be revealed by
autoradiography of the nitrocellulose disc. Bacterial colonies
(phage plaques) containing clones bearing target DNA are
identified on the film and can be recovered from the master 357
plate.
• Host bacteria transformed with a plasmid-based genomic library or
infected with a bacteriophage-based genomic library are plated on
a petri plate and incubated overnight to allow bacterial colonies (or
phage plaques) to form. A replica of the bacterial colonies (or
plaques) is then obtained by overlaying the plate with a
nitrocellulose disc (1). Nitrocellulose strongly binds nucleic acids;
single-stranded nucleic acids are bound more tightly than double-
stranded nucleic acids. (Nylon membranes with similar nucleic acid–
and protein-binding properties are also used.) Once the
nitrocellulose disc has taken up an impression of the bacterial
colonies (or plaques), it is removed and the petri plate is set aside
and saved. The disc is treated with 2 M NaOH, neutralized, and
dried (2). NaOH both lyses any bacteria (or phage particles) and
dissociates the DNA strands. When the disc is dried, the DNA
strands become immobilized on the filter. The dried disc is placed in
a sealable plastic bag, and a solution containing heat-denatured
(single-stranded), labeled probe is added (3). The bag is incubated
to allow annealing of the probe DNA to any target DNA sequences
that might be present on the nitrocellulose. The filter is then
washed, dried, and placed on a piece of X-ray film to obtain an
autoradiogram (4). The position of any spots on the X-ray film
reveals where the labeled probe has hybridized with target DNA (5).
The location of these spots can be used to recover the genomic
clone from the bacteria (or plaques) on the original petri plate. 358
359
Colony hybridization for screening of DNA library

360
Using a DNA
probe to identify
a cloned gene in
a population of
bacteria

361
Screening by Colony Nucleic Acid
Hybridization

362
Southern blotting technique screening:
• Experimentally, the gene library to be screened, or the
DNA bands separated by electrophoresis on an agarose
gel, is transferred to a nitrocellulose or nylon filters by
blotting. These are then processed to release DNA from
the bacteria and/or the phage. These are then
denatured to separate the complementary strands and
are immobilized on to the membrane, in the position
occupied by the original clone and in a way that leaves
the bases free to interact with the complementary
molecules. The probe molecules are then labeled
(traditionally with radioactive nucleotides but now more
commonly with chromogenic or chemiluminescent
labels). Probe and filter are then incubated under
conditions that promote hybridization after which the
unbound probe is washed off from the filter and the
specifically bound probe is visualized. Positive signals
reveal the position (and thus identity) of those
recombinants carrying the sequences related to the
probe. 363
Southern blotting to screen DNA library or the recombinants containing
gene of interest

364
Preservation of DNA Libraries:
• DNA libraries need to be preserved for future use and
analysis, as they represent a valuable resource of genetic
information.
• The preservation of DNA libraries typically involves the
following methods:
• Freezing in Glycerol Stocks: Bacterial or yeast cultures
containing the DNA library are mixed with glycerol and stored at
ultra-low temperatures, usually -80°C or in liquid nitrogen.
• Store DNA libraries at -20°C or -80°C for long-term preservation. For
short-term use, 4°C may be acceptable
• Cryopreservation: Bacterial or yeast cells containing the DNA
library are mixed with cryoprotectants (substances that protect
biological materials, such as cells, tissues, and organs, from damage
during freezing and thawing processes. E.g. Glycerol, Mannitol,
Dimethyl sulfoxide, Proline, etc.) and slowly frozen to preserve the
viability of the cells and the DNA library.
• Lyophilization (Freeze-drying): The DNA library is dried under
vacuum, often in the presence of a protective agent, and stored at
low temperatures.
• The choice of preservation method depends on factors such
as the type of library, the host organism, the intended use of
the library, and the available resources and facilities. 365
Chapter 5

Gene transfer techniques

1. Biological Methods (e.g. Agro-
Bacterium Mediated Gene
Transfer in Plants)
2. Chemical Methods and
3. Physical/Mechanical Methods

366
Gene Transfer Techniques:
• Gene transfer techniques refer to the methods used
to introduce foreign genes into host organisms,
which is a crucial part of genetic engineering and
biotechnology. There are various techniques for gene
transfer, which can be classified into physical,
chemical, and biological methods.

Applications of Gene Transfer Techniques

• Research:
• Understanding gene function and regulation.
• Medicine:
• Gene therapy for genetic disorders and cancer treatment.
• Agriculture:
• Development of genetically modified organisms (GMOs) for
improved traits.

367
Common Gene Transfer Techniques:
A) Biological Methods
1. Agrobacterium-Mediated Transformation:
• A biological method where the bacterium Agrobacterium
tumefaciens transfers a segment of its DNA (T-DNA) into plant
cells.
• Example: Commonly used for generating transgenic plants.
2. Viral-Mediated Gene Transfer:
• Utilization of viruses to deliver foreign DNA into host cells. Viruses
naturally infect cells and integrate their DNA, making them
efficient gene delivery vectors.
• Example: Adenovirus or lentivirus used in gene therapy to deliver
therapeutic genes into human cells.
3. Conjugation:
• The direct transfer of DNA from one bacterium to another through
cell-to-cell contact.
• Example: Transfer of plasmid DNA between bacterial cells.
4. Transformation:
• The uptake of naked DNA by a host cell (commonly bacteria).
• Example: E. coli uptake of plasmid DNA during cloning
experiments. 368
B) Physical/Mechanical Methods
5. Microinjection:
• Direct injection of DNA into the nucleus of a cell using a fine needle.
• Example: DNA microinjection in fertilized animal eggs for the generation of
transgenic animals.
6. Electroporation:
• A physical method where an electric field is applied to cells, making the cell
membrane more permeable to DNA.
• Example: Inserting plasmids into bacteria or eukaryotic cells using
electroporation.
7. Biolistics (Gene Gun):
• A physical method that shoots microscopic gold or tungsten particles coated
with DNA into cells.
• Example: Gene transfer into plant cells to create transgenic crops.

C) Chemical Methods
8. Liposome-Mediated Gene Transfer:
• Encapsulation of DNA in lipid-based vesicles (liposomes) that can fuse with
the host cell membrane to deliver DNA.
• Example: Lipofection used for introducing DNA into mammalian cells.
9. Transfection:
• The introduction of foreign DNA into eukaryotic cells, commonly using
chemicals or physical methods like electroporation.
• Example: Transfecting mammalian cells with plasmids to express 369

recombinant proteins.
A) Biological Method
Agrobacterium-mediated gene
transformation
 among vector dependant gene transfer methods,
Agrobacterium-mediated genetic transformation is
most widely used for the expression of foreign genes
in plant cells.
Agrobacterium tumefaciens
 is a soil borne bacterium
 rightfully called the “nature’s most
effective plant genetic engineer”.
 very efficient, but limited to a selected
group of plants. i.e restrict to only
dicotyledonous plants.
 cause crown gall tumors (disease)
 contain the plasmid that is responsible for 370
Ti plasmid (tumor-inducing plasmid)
- has three important regions
1. T-DNA region
2. Virulence region
3. Opine metabolizing region

 The region of the Ti plasmids responsible for

tumor formation is known as the T-DNA 371
Steps of T-DNA transfer & integration into plant genome

1. Signal induction to Agrobacterium by phenolic

compounds released by wounded plant cells
2. Attachment of Agrobacterium to plant cells
3. Production of virulence proteins
4. Production of T-DNA strand
5. Transfer of T-DNA into plant cells
6. Integration of T-DNA into plant genome

372
 373
Natural plant genetic engineering by Agrobacterium tumefaciens
374
375
376
Ti plasmid used as a vector for DNA
delivery in plant cells
• Ti plasmid can be disarmed by removing
the T-DNA parts keeping the borders intact
and can also be used as vectors. Genes of
interest can be cloned in place of T-DNA,
and agrobacterium can now introduce this
foreign DNA into the plant gene mistaking it
for T-DNA.

377
Modified Ti plasmid used as a vector for
DNA delivery in plant cells to produce
Transgenic plants

378
379
380
The Ti plasmid can be used to transfer genes into plants.
Flanking sequences TL and TR are 381
• Explanation of the above figure: The soil bacterium
Agrobacterium tumefaciens, which invades plants through
wounds and induces crown galls (tumors), has been used
to transfer genes to plants. This bacterium contains a large
plasmid called the Ti plasmid, part of which is transferred
to a plant cell when A. tumefaciens
infects a plant. In the plant, part of the Ti plasmid DNA
integrates into one of the plant chromosomes, where it is
transcribed and translated to produce several enzymes
that help support the bacterium (Figure a). Geneticists
have engineered a vector that contains the flanking
sequences required to transfer DNA, a selectable marker,
and restriction sites into which foreign DNA can
be inserted (Figure b). When placed in A. tumefaciens
with the Ti plasmid, this vector will transfer the foreign
DNA
that it carries into a plant cell, where it will integrate into a
plant chromosome. This vector has been used to transfer
genes that confer economically significant attributes such
as
resistances to herbicides, plant viruses, and insect pests.
382
The use of Agrobacterium tumefaciens as a gene
transfer technique in Plants

383
B) Physical/Mechanical Methods
(Direct Gene Transfer Techniques)

• One of the major problems in agrobacterium-

or vector-mediated gene transfer is the host
specificity. i.e. Agrobacterium cannot infect
most of monocot plants.
• Therefore, alternate gene transfer
techniques should be developed, which
don’t involve infection by any organism.
• The following are some of the direct gene
transfer methods developed for the genetic
engineering of specific plants:

384
1. Microinjection
• The desired gene in the form of plasmid or alone is
injected directly into the plant protoplast. The
DNA may be directly delivered into the nucleus or in
the cytoplasm. Injection into the nucleus is more
efficient than that into the cytoplasm.
• is carried out with automatic equipments
(robotics) using a micro-needle. Micro-injection,
however, has produced only a few transgenic
plants. The technique is laborious, technically
difficult, and limited to the number of cells
actually injected.
• Direct injection of DNA into the nucleus of a cell (if it is animal
cell) using a fine needle. Commonly used in the generation of
transgenic animals

385
2. Electroporation
• This is a physical technique developed for DNA
delivery into plant protoplasts through
membranes under a rapid pulse of high-
voltage direct current. When an electric pulse
of high voltage is applied in the presence of
protoplasts, it makes a large number of
temporary pores in the cell membrane. These
temporary openings facilitate the DNA uptake by
the protoplasts. Once inside the cell, the DNA is
integrated and the foreign gene will express.
• Electroporation= Introducing DNA or
chromosomes into cells using an electric pulse to
create temporary pores in the cell membrane.

386
Electroporation

Mixture of
plant cells
& Vectors

387
3. Biolistic Method (Microprojectile or
Particle-gun Method)
• This is a new method that involves
accelerating very small particles of tungsten
or gold coated with DNA into cells using an
electrostatic pulse, air pressure, or gunpowder
percussion. The DNA molecules containing
the desired gene are coated on to
microscopic gold or tungsten particles.
As the particles pass through the cell, the DNA
dissolves and becomes free to integrate into
the plant-cell genome.
• the commonest (a widely used and
highly successful) method of
transfecting plants
• in this method a so called gene gun or 388
• Biolistics has the advantage of being
applicable to whole cells in suspension or to
intact or sliced plant tissues. For example,
plant meristems or tissues capable of
regeneration can be targeted directly.
• i.e. It is a widely used and highly successful
method of transfecting plants using plant
protoplasts, plant cell suspensions, callus
cultures, even chloroplasts and mitochondria,
and indeed any form of plant preparation
capable of regeneration in dicots, monocots,
and conifers.
• Unlike electro-poration and microinjection,
this technique does not require protoplasts
or even single-cell isolations.
389
C) Chemical-mediated Gene Transfer
Techniques
• They are also Direct Gene Transfer Techniques
• Protoplasts will take up pure DNA when
treated with certain membrane-active agents
such as:
• CaCl2(makes pores in cell membrane)
• Polyethylene glycol (PEG), and
• Dextran sulphate
• Calcium phosphate,
• Lipofection (liposomes), or
• Polyethylenimine (PEI)
• Once inside the cell, the DNA is integrated
into the genome and the foreign gene will
express. This technique largely depends on
390
Chapter 6
Transgenic Science and Genetic improvement

 Transgenic Science in Plant

Improvement and Bio pharming
 Transgenic Science for Animal
Improvement and Bio pharming
 Gene Mapping in Plants and
Animals
 Other applications

391
Transgenic Science: An Overview
• Transgenic science involves the introduction of foreign DNA
into an organism’s genome, creating a genetically modified
organism (GMO) with new or altered traits. This field is a branch of
genetic engineering and has revolutionized agriculture, medicine,
and research by enabling the creation of organisms that exhibit
traits not naturally present in the species.
Key Concepts in Transgenic Science:
1.Transgene
• The foreign gene that is inserted into the genome of the host
organism. This gene can originate from a different species, making
the organism transgenic.
2.Genetic Modification
• The process of altering the genetic makeup of an organism through
biotechnology. Transgenic organisms have been genetically
modified to contain and express genes from other species.
3.Vector
• A tool used to introduce the transgene into the host organism.
Common vectors include:
• Plasmids, Viruses and Phages
4.Gene Expression
• After insertion, the transgene is expressed, leading to the 392
production of a specific protein that confers the new trait.
Applications of Transgenic Science
1. Agriculture
• Pest-Resistant Crops: Plants like Bt cotton and Bt corn are engineered to produce
a toxin derived from the bacterium Bacillus thuringiensis (Bt), which is harmful to
insects but safe for human consumption.
• Herbicide-Resistant Crops: Crops like Roundup Ready soybeans are engineered
to tolerate specific herbicides, allowing farmers to control weeds without harming
the crop.
• Nutritionally Enhanced Crops: Example includes Golden Rice, which is
engineered to produce beta-carotene, a precursor to vitamin A, helping to combat
vitamin A deficiency in developing countries.
2. Medicine
• Gene Therapy: Transgenic techniques are used to introduce healthy copies of
defective genes into patients' cells, offering potential cures for genetic disorders
such as cystic fibrosis and hemophilia.
• Biopharmaceuticals: Transgenic animals and plants are used to produce
therapeutic proteins. For instance, goats have been engineered to produce
antithrombin, a protein used to prevent blood clots, in their milk.
• Edible Vaccines: Plants like potatoes and tomatoes are being developed to
produce vaccines that could be consumed, offering a cost-effective and needle-free
vaccination method.
3. Research
• Model Organisms: Transgenic animals, such as mice, are used as models to study
human diseases, drug effects, and gene functions. Knockout mice, where specific
genes are "knocked out," help researchers understand gene roles.
• Functional Genomics: The study of gene function using transgenic organisms393
Steps in Creating a Transgenic Organism
1.Identification of the Target Gene: The gene of interest
(transgene) is identified for the desired trait or function.
2. Cloning the Gene: The transgene is cloned into a
suitable vector, such as a plasmid, ensuring it can be
efficiently introduced into the host organism.
3. Gene Transfer: The vector is introduced into the host
organism’s cells through various techniques:
4. Selection of Transgenic Cells: Cells that have
successfully incorporated the transgene are identified and
selected for further growth and development.
5. Regeneration of the Transgenic Organism: For
plants, transgenic cells are grown into full plants using
tissue culture techniques. For animals, transgenic embryos
are implanted into surrogate mothers to develop into
transgenic offspring.
6. Verification and Expression: The presence and
expression of the transgene are confirmed through
molecular techniques such as PCR, Southern blotting, 394 or

Western blotting.
Benefits and Challenges of Transgenic Science
A) Benefits:
1. Improved Crop Yields: Genetic modifications help
crops resist pests, diseases, and environmental
stresses, leading to higher yields.
2. Nutritional Enhancement: Crops can be fortified
with essential nutrients, addressing malnutrition in
developing countries.
3. Disease Resistance: Transgenic animals can be
engineered to resist specific diseases, reducing losses
in livestock farming.
4. Pharmaceutical Production: Transgenic plants
and animals provide a cost-effective means of
producing medicines and vaccines.
5. Scientific Advances: Transgenic organisms are
invaluable tools in understanding gene function,
disease mechanisms, and developing new medical 395
treatments.
B) Challenges:
1. Ethical Concerns: The creation of GMOs raises
ethical questions about tampering with nature,
animal welfare, and the potential impact on
biodiversity.
2. Environmental Impact: The release of
transgenic organisms into the environment could
have unintended consequences, such as gene flow
to wild populations or the development of
resistance in pests.
3. Public Perception and Safety: Many people
are concerned about the safety of GMOs in the food
supply, leading to regulatory and labeling debates.
4. Intellectual Property Issues: Patenting
genetically modified organisms has sparked legal
and ethical debates over the ownership of life
forms and access to biotechnological innovations.
396
Transgenic Science for Plant
Improvement and Biopharming
• Transgenic technology allows for the
introduction of foreign genes into plants to
enhance desirable traits such as yield,
resistance to pests, diseases, herbicides, or
environmental stresses like drought and
salinity. These genetically modified plants
have transformed agricultural practices,
leading to higher productivity and food
security.

397
Key Applications of Transgenic Science in
Plant Improvement:
1.Herbicide Resistance:
• Example: Roundup Ready soybeans are engineered
to be resistant to glyphosate herbicides, allowing
farmers to control weeds without harming crops.
2. Insect Resistance:
• Example: Bt crops (e.g., Bt cotton, Bt corn) are
engineered to express the Bacillus thuringiensis (Bt)
toxin, which is harmful to insect pests but safe for
humans and other animals.
3. Disease Resistance:
• Transgenic plants can be engineered to resist viral,
bacterial, or fungal infections.
• Example: Papaya plants have been modified to resist
the papaya ringspot virus.
398
4. Improved Nutritional Content:
• Genetic engineering can enhance the nutritional
profile of crops.
• Example: Golden Rice is enriched with provitamin A
(beta-carotene) to address vitamin A deficiency in
developing countries.
5. Drought and Salinity Resistance:
• Transgenic plants are being developed to survive in
harsh environmental conditions like drought, high
salinity, or extreme temperatures.
• Example: Drought-tolerant maize is designed to
improve yields in regions with limited water
availability.
6. Delayed Ripening:
• Modification of ripening-related genes allows for
extended shelf life of fruits and vegetables.
• Example: Flavr Savr tomato, engineered for delayed
ripening to reduce spoilage. 399
Transgenic Science in Plant
Biopharming
The (Bioreactors)
most important reasons for developing
transgenic plants are:
1. To develop plant bioreactors for inexpensive
manufacture of commercially important
products. i.e. Transgenic Plants used as
Bioreactors (Biopharming).
• Biopharming refers to the production of
pharmaceutical compounds in genetically
modified plants, providing a cost-effective and
scalable method to produce vaccines, antibodies,
and other therapeutic proteins.
• Therefore, if the plants are transformed with
the right kind of gene, they can function like a
Biopharming (bioreactor) to produce the
required compounds. 400


• Example of Plant-Based Biopharming: Plantibody
production: Tobacco plants have been engineered to
produce monoclonal antibodies used to treat various
diseases.
• Edible vaccines: Plants such as potatoes and bananas
are being developed to produce vaccines that can be
consumed, offering an alternative to traditional vaccine
delivery methods.
• Diagnostic and therapeutic Proteins (plasma
proteins, peptide hormones, interferons, and
cytokinins)
• Recombinant vaccines (e.g. Edible vaccines)
• Secondary metabolites (alkaloids, flavanoids,
tannins, terpins, essential oils, latex, etc)
2. To improve agricultural, horticultural, or
ornamental value of plants
3. To study the action of genes in plants during
development and various biological processes 401
Transgenic Science for the development of
Insect (pest) resistance plant
• The first insect-resistant genes used for
making insect resistant crops were the Cry
genes or the Bt genes isolated from
Bacillus thuringiensis, a pathogen that
kills certain group of insects.
• The latest example of cultivating an insect-
resistant crop is the Bt cotton plant, the
cotton plant transformed with the Bt toxin
genes, the Cryl A(c) gene. It is resistant to
the ball worm (helicoperpa armigera), which
causes great damage to cotton cultivation
all over the world.
403
404
Transgenic Science for the development of
Fungal and Bacterial resistance plant
Gene that encodes enzyme . E.g. Chitinase or Lysozyme

Introduction of the gene into the plant that encodes

enzyme chitinase and Lysozyme.
Plant infection by Fungi
or Bacteria

Degradation of Chitin or Peptidoglycan

No survival of Fungi and Bacteria in the plants

405
Normal plants
Transgenic Science for the development
of plants that are resistance to abiotic
stress plants (e.g. herbicide, drought, salinity, etc.
resistance plant)
a) Herbicide Resistance Plants
Weeds account to 10– 15% of the reduction in crop
yield. The growth of weeds is controlled by applying
herbicides to the field. However, the herbicides do
not discriminate between the weeds from the crop
plant. Therefore, it is important to develop plants,
which are tolerant to the action of herbicides.
Glyphosate is a broad spectrum herbicide
effective against worst weeds. Soil inhabiting
microbes have the ability to detoxify glyphosate.
These microbes possess a gene (gox gene) that
codes for glyphosate oxidase, which converts
glyphosate to glyoxalate and
406
 Herbicide resistance plants can be
produced by
i) by detoxification of herbicide by using a foreign
gene which encode the chemical that can neutralize
the herbibcide. E.g. Glyphosate oxidase from
microorganism.

(Glyphosate)

407
ii) by over expression of the target protein which is affected by herbicides. i.e. by integrating multiple
copies of the gene which can encode the protein (enzyme)

(Glyphosate)

Because target protein is

affected by herbicides
408
409
Transgenic Science for the development
Drought, salinity, etc. resistance plants
• Plants that are capable of withstanding climate
changes are likely to produce greater yields
during severe weather conditions.
• There are metabolites produced in plants due
to the stress response of high temperature,
drought, or salinity.
• These metabolites include stress-related
osmolytes such as sugars and sugar derivatives
such as trahalose, fructans, mannitol, etc., and
amino acids such as proline, glycine, betaine,
and proteins such as heat-shock proteins and
anti-freeze proteins. Crop plants have been
genetically engineered to over-express genes
responsible for the production of one or more of
the above-mentioned metabolites. Such plants
are found to show increased stress tolerance.
Transgenic Science for the
development of quality
enhancing plant varieties
e.g To increase the shelf life of fruits and
vegetables

• Delayed-Ripening Fruits and Vegetables

This can be achieved by anti-sense mRNA

technology

411
Genetic regulation of fruit ripening. An antisense copy of the gene for ethylene biosynthesis
prevents the formation of ethylene and subsequent ripening of transgenic fruit. The antisense strand is
complementary to the sequence for the ethylene biosynthesis gene. After transcription, the antisense
mRNA pairs with the sense mRNA, and the doublestranded mRNA cannot be translated into a functional
protein. Ethylene is not produced, and the fruit does not ripen. The fruit is sturdier for shipping
412 in its
unripened form and can be ripened later with exposure to ethylene. Thus, while wild-type tomatoes
Transgenic Science for the
development of nutritionally
improved foods
a. Improving essential amino acids in cereal crops
• Some crops that are improved to
provide essential amino acids
• Rice: lysine
• Corn: lysine and methionine
• Potato: lysine and methionine
• etc.
b. improving the Vitamins and minerals of
cereal crops
• E.g. “ golden rice” is improved for Vitamin A
Transgenic Science for Animal Improvement and
Biopharming
• Transgenic Science for Animal Improvement: Transgenic
animals are genetically engineered to express desired traits or to
serve as models for human diseases. This technology has a
significant impact on agriculture, medicine, and scientific
research.
Key Applications of Transgenic Science in Animal
Improvement:
1.Increased Growth Rate:
• Genetic modifications can enhance growth rates, leading to increased
productivity in livestock.
• Example: AquAdvantage salmon grow faster than conventional salmon due
to the insertion of a growth hormone gene from Chinook salmon.
2. Disease Resistance:
• Animals can be engineered to resist infectious diseases, improving animal
welfare and reducing the use of antibiotics.
• Example: Pigs engineered to resist porcine reproductive and respiratory
syndrome (PRRS), a common viral infection in pigs.
3. Improved Milk Production and Composition:
• Genetic modifications can enhance milk yield and its nutritional composition.
• Example: Cows have been genetically modified to produce milk with less
lactose or enriched with specific proteins for human consumption. 415
Applications of Transgenic Science
in animal Biopharming
(Bioreactors)
E.g. to produce medically useful proteins.
• transgenic cattle were created to
produce milk containing particular
human proteins, which may help in
the treatment of human disease.
• Therefore, the advantage with transgenic
animals is to produce scarce human
proteins in huge quantities.
• Thus, the animals serving as factories for
production of biologically important
products are referred to as
• animal bioreactors or sometimes
416

pharm animals.
Hence, similar to plants, transgenic animals are used for
biopharming to produce therapeutic proteins, antibodies, or
other pharmaceutical products. The use of animals allows for
complex protein production that may not be feasible in plants
or microbes.
Some transgenic animals that serve as Biopharming
(bioreactors) are:
 Transgenic cow
• for the production of lactoferrin and interferons.
 Transgenic goat
• to synthesize tissue plasminogen activator, and antithrombin
III
• For production of anti-thrombin: Goats have been genetically
modified to produce the anticoagulant protein anti-thrombin in
their milk, which is used to prevent blood clots.
 Transgenic mouse
• for the production of immunoglobulins, and urokinase.
 Transgenic chickens
• to produce proteins in their eggs that can be harvested and
used for medicinal purposes used as a pharmaceuticals.
 Transgenic pig
Transgenic animals as model
animals
 to understand, and treat human
diseases
e.g. Transgenic mice/ Knockout mice
• They are good models for many human
diseases, such as
• Alzheimer disease, and are useful as test systems to
evaluate potential therapies.
• They provide important information about the
consequences of defective gene products, the
course of the disease, and the effectiveness of
different therapies.
• E.g. The XenoMouse or the Harvard mouse,
• which synthesizes completely human antibodies (human
heavy and light antibody chains).
• They have also been used to produce other
Transgenic animals as biosensors of
environmental pollutants
 E.g. Transgenic fish
 Transgenes encoding fluorescent proteins
under the control of promoters that are
activated when particular contaminants,
such as estrogenic compounds, are present
have been incorporated into fish for
determining the presence of pollutants in
water.
Transgenic animals used to study
gene function and regulation
• Gene Mapping in Plants and Animals
• Gene mapping is a fundamental technique in genetics
that involves identifying the location of genes on
chromosomes. It provides insights into the organization
and function of genes, facilitating a deeper understanding
of inheritance, genetic diseases, and the evolution of
species.
Several techniques have been developed for gene
mapping:
 Genetic linkage mapping: This method analyzes the
inheritance patterns of traits to determine the relative
distances between genes on a chromosome. It is based
on the principle that genes located close together on a
chromosome are more likely to be inherited together.
 Physical mapping: This involves using molecular
biology techniques to determine the physical location of
genes on a chromosome. It can be achieved through
420
methods such as restriction enzyme mapping and
Applications of Gene Mapping
 Disease gene identification: Genetic mapping can
help identify genes responsible for inherited diseases,
leading to improved diagnosis and treatment.
 Agricultural improvement: Breeders can use
genetic mapping to select plants with desirable traits,
such as higher yield, disease resistance, or improved
nutritional value.
 Understanding genetic diversity: Genetic
mapping can help study the genetic variation within
and between species, providing insights into
evolution and adaptation.
 Pharmacogenomics: Genetic mapping can be used
to identify genetic factors that influence drug
response, leading to personalized medicine. 421
Chapter 7
Microbial biotechnology: genetic manipulation

• Microbial biotechnology is a field that

utilizes microorganisms to produce valuable
products or perform specific functions.
• Genetic manipulation is a cornerstone of
microbial biotechnology, allowing scientists
to modify the genetic makeup of
microorganisms to enhance their
capabilities or create novel phenotypes.
422
Key Techniques for Genetic Manipulation:
 Recombinant DNA technology: This involves
combining DNA from different sources to create new
genetic constructs. It involves techniques like
restriction enzyme digestion, ligation, and
transformation.
 Transformation: The introduction of foreign DNA
into a host cell. This can be achieved through
methods like electroporation, chemical
transformation, or natural competence.
 Transduction: The transfer of DNA between
bacterial cells mediated by a bacteriophage.
 Conjugation: The transfer of genetic material
between bacterial cells through direct contact.
 Genome editing: Precisely modifying specific DNA
423
sequences using techniques like CRISPR-Cas9.
Applications of Genetic Manipulation in Microbial
Biotechnology (sector based application)
1. Medicine
 Production of therapeutic proteins (e.g., insulin, monoclonal
antibodies).
 Development of gene therapies for genetic disorders.
•2. Agriculture
 Genetically modified organisms (GMOs) for pest resistance and
increased yield.
 Biopesticides and biofertilizers to enhance crop productivity.
3. Environmental Biotechnology
 Bioremediation: Using microbes to clean up contaminated
environments.
 Waste treatment and bioenergy production (e.g., methane from
anaerobic digestion).
4. Industrial Biotechnology
 Production of enzymes for food, textiles, and biofuels.
 Bioprocessing for sustainable manufacturing. 424
Applications of Microbial Genetic Manipulation
(product based application):
 Production of biopharmaceuticals:
 Microorganisms can be engineered to produce therapeutic
proteins such as insulin, growth hormones, and vaccines.
 Bioremediation:
 Genetically modified microorganisms can be used to degrade
pollutants and clean up contaminated sites.
 Biofuels:
 Microorganisms can be engineered to produce biofuels like
ethanol and biodiesel.
 Food production:
 Genetically modified microorganisms can be used to improve the
nutritional content or shelf life of food products.
 Industrial enzymes:
 Microorganisms can produce enzymes for various industrial
applications, such as food processing, detergents, and biofuels.
425
Chapter 8
Gene Therapy
• Introduction to Gene Therapy
• Methods, Strategies,
Approaches, and Types of
Gene Therapy
• Gene Targeting & Silencing
• Gene Therapy in the
Treatment of Diseases
• Challenges and Future of
Gene Therapy
426
Gene therapy
• is the process of treating a genetic disease by
replacing a person’s defective gene with a new,
normally functioning one.
• is a therapeutic technique that involves the
introduction of genetic material (DNA or RNA) into
cells to treat or prevent diseases. It aims to correct
defective genes or introduce new genes to treat
disorders at the molecular level.
• It is the process by which replacement of defective
gene/s with normal ones to cure the diseases.
Including:
e.g. Cystic fibrosis,
Sickel cell anemia
Hemophilia,
Neurological diseases
Cancer,
Severe Combined Immuno Deficiency 427
Historical background of Gene therapy
• Gene therapy has a much shorter history than
genetic engineering, dating back only to 1990,
when doctors performed a ground breaking
procedure on four-year-old Ashanthi DeSilva.
DeSilva suffered from severe combined
immunodeficiency (SCID), which meant her
immune system lacked the ability to fight off even
mild infections. People born with SCID typically die
in childhood. Researchers took some of DeSilva’s
white blood cells, grew them in a laboratory,
inserted a missing gene into them, and then put
the modified cells back into her bloodstream.
While the treatment was not a cure, it did boost
DeSilva’s immune system enough for her to lead a
normal life, although she must receive treatment
every few months as the modified cells begin to
die off and need to be replaced 428
• The success of DeSilva’s gene therapy encouraged scientists, but
their optimism proved short-lived. The next major clinical trial of
gene therapy in 1999 ended in the death of 18-year-old Jesse
Gelsinger, who was afflicted with ornithine transcarbamylase
deficiency, a liver disease that usually results in death at birth.
Gelsinger’s version of the disease, in which his liver could not
metabolize ammonia, resulted from a genetic mutation rather than
hereditary factors, and he had survived on a strict diet and regimen
of drugs until he underwent gene therapy. Doctors at the University
of Pennsylvania created an adenovirus that contained a corrected
gene and injected it into Gelsinger. his body initiated a massive
immune response to the virus used to
insert the new gene, which led to organ failure and then death five
days later. The trial was a huge setback for gene therapy
researchers.
• Researchers have high hopes for developing gene therapies for
single gene diseases, such as cystic fibrosis, hemophilia,
muscular dystrophy, and sickle cell anemia. Progress is slow
because of the size of the human genome; finding where
exactly to insert a modified gene is a daunting task, as is finding
an appropriate vector, or carrier. Viruses are often used as
vectors; modified genes are inserted into the virus, which is
then introduced in the patient with the hope that it will infect
the target cell with the altered gene. 429
Approaches for gene therapy (i.e.
in which cells or organs gene
therapy could performed)
1.Somatic cell gene therapy
• gene therapy in non-reproductive
cells
2.Germ cell gene therapy
• gene therapy in reproductive cells

430
1.Somatic cell gene therapy
• Transfer of a gene or genes into body cells
(somatic cells) other than germ cells
(reproductive cells) with effect only on the
patient.
• In the case of somatic cell gene therapy
therapeutic genes are transferred into the
somatic cells, or body cells, of a patient.
Any modifications and effects will be
restricted to the individual patient only, and
will not be inherited by the patient's
offspring or later generations. I.e. The new
genetic material cannot be passed on to
offspring. i.e. the genetic alternation is not
observed or not transmitted to the
successive generations. 431
Example of somatic cell gene therapy
 Gene therapy for Cystic fibrosis
• Cystic fibrosis is a disease characterized by
production of thick and viscous lung mucous in
the lung. The mucous makes breathing very
difficult and, in many cases, is fatal.
- a genetically engineered virus, carrying the
corrective gene, introduced into the patient's
lung cells would allow the lungs to function
properly. Because the introduced gene
(functional gene) would allow the lung cells to
produce a protein that eliminates the mucus.

432
Example of somatic cell gene therapy: Gene therapy for Cystic fibrosis

433
 Gene therapy for a patient of Severe
Combined Immuno Deficiency (caused by
the deficiency of the enzyme adenosine
deaminase)
• SCID is rare inherited immune disorder associated
with T-lymphocytes, and B-lymphocytes
dysfunction. SCID patients have a defect in the that
encodes for adenosine deaminase. In the deficiency
of ADA, deoxyadenosine and its metabolites
accumulate and destroy T-lymphocytes. T-
Lymphocytes are essential for body’s immunity.
Besides participating directly in body’s defense,
they promote the function of B-lymphocytes to
produce antibodies. Thus, the patients of SCID
(lacking ADA) suffer from infectious diseases
and die at an young age.
434
Treatment of Adenosine Deaminase
(ADA) deficient patient by somatic ex
vivo gene therapy (SCID-Severe
combined immunodeficiency).

435
2. Germ cell gene therapy (gene therapy in
reproductive cells)
• Germ-line cell therapy involves the introduction of
corrective genes into reproductive cells (sperm and eggs)
or zygotes, with the objective of creating a beneficial
genetic change that is transmitted to the offspring.
• In this approach, the introduction of DNA in to germ cells
(sperm or eggs) is passed on to the successive
generations. When genes are introduced in a
reproductive cell, descendant cells can inherit the genes.
• Highly effective in counteracting genetic disorders and
hereditary diseases.
• However, Germ-line gene therapy is presently not being
pursued because it is technically extremely difficult and
is ethically and socially unacceptable.
• Therefore, for safety, ethical and technical reasons, germ
cell gene therapy is not being attempted at present.

436
Based on the therapy takes place outside the cell
or inside the cell, gene therapy can be divided in
two types.
There are two main strategies of gene therapy:
in vivo and ex vivo
I. In vivo gene therapy
• the target gene (therapeutic gene) is
delivered directly into the patient's body,
targeting specific cells or tissues.
• more or less random process;
• small ability to control; less manipulations.
• only available option for tissues that can not
be grown in vitro; or if grown cells can not be
transferred back.
437
II. Ex vivo gene therapy
• Involves the transfer of genes in cultured cells (e.g. bone
marrow cells) which are then reintroduced in to the
patient body.
Steps of Ex vivo gene therapy
• First the targeted cells are removed or isolated from the
patients body
• Then the genetic material (therapeutic gene) is transferred
into the cells grown in vitro (laboratory). i.e. delivery takes
place out of the body
• Genetically modified cells are selected and expanded; more
manipulations
• Finally, genetically modified cells are then returned back
(placed back) to the patient’s body.
• Genetically, in case of Ex vivo gene therapy cells are
manipulated out of the body and then reimplanted into
the patient
• It is controlled process
• Ex vivo gene therapy is costly manipulated but it is
highly efficient to transfer target gene in to target
438
 1
2
4

3
in vivo gene therapy // ex vivo gene therapy

440
Vectors used for gene delivery in gene therapy
• Viruses - retroviruses are frequently used for
gene therapy as a vector e.g. HIV.
 How viruses can be used as a vector for gene
therapy? Ans: by making them harmless.
 How they can be harmless?
Ans: by removing a gene that encodes for the
viral envelope. This is because, without the
envelope, retrovirus cannot enter the host cell.
• Bone marrow cells- because they contain
totipotent embryonic stem cells. These are
capable of dividing and differentiating into
various cell types, and they are the potential
candidate for gene therapy of sickel cell anemia.

• Human artificial chromosome 441

Human Gene Therapy using
Retroviruses

Use of a
Retrovirus for
Gene Therapy

Reverse transcriptase
convert RNA to DNA in
the cell

442
How vectors work in gene therapy?
• A vector delivers the therapeutic gene into a
patient’s target cell
• The target cells become infected with the
viral vector
• The vector’s genetic material is inserted into
the target cell
• Functional proteins are created from the
therapeutic gene causing the cell to return
to a normal state
• Which one of the following is frequently used as a
vector during gene therapy? Why? explain your
reason
A. Bacteria
B. Fungi
C. Viruses
D. Agrobacterium tumefaciens
E. Bacillus thuringienesis
F. All
• Because Viruses are cell, tissue and organ specific and
they are also intercellular parasites. Which means that
they have the ability to targeting the therapeutic gene in
specific location. Generally, most viruses can integrate
their genetic material in the host cell.
• Due to the ability of viruses to infect a wide variety of
cells, they are one of the most efficient vectors for gene
transfer into the target cells.
e.g. Retrovirus vectors integrate stably into host
chromosome.
How Gene Therapy can be performed?
Using:
1. Gene Replacement (gene augmentation):
Involves inserting a normal copy of a gene to
replace a mutated one.
2. Gene Editing: Techniques like CRISPR-Cas9
enable precise modifications of the genome.
3. Gene Silencing (gene inhibition): Involves
reducing or eliminating the expression of a gene
through RNA interference (RNAi) or antisense
oligonucleotides.
4. Viral Vectors: Utilizes modified viruses to
deliver therapeutic genes into target cells.
5. Non-viral Delivery: Methods such as
electroporation, liposomes, and nanoparticles
facilitate gene transfer without using viruses.
445
How gene therapy is achieved or
performed?
1.By gene augmentation therapy- i.e. in
this strategies a DNA is inserted into the
genome to replace the missing gene
product

446
2. By gene inhibition therapy- i.e. the
antisense gene inhibits the expression of
the dominant gene.

447
Gene Targeting & Silencing
• Gene targeting
• refers to the deliberate modification of a specific gene within an
organism's genome. This involves introducing a specific DNA
sequence to replace or disrupt a defective gene. Techniques like
homologous recombination and CRISPR-Cas9 (gene-editing
technology)are commonly used for gene targeting.
• Homologous Recombination:
• Used in embryonic stem cells to integrate a new gene at a specific
location, often for creating knockout model animals.
• CRISPR-Cas9: Gene-editing technology
• Allows for targeted modifications, including insertions, deletions, or
replacements.
• Gene Silencing:
• refers to inhibition of the expression of a gene
• i.e. Gene silencing aims to reduce or completely inhibit the
expression of specific genes.
Common methods include:
• RNA Interference (RNAi):
• is a powerful technique for gene silencing, using small RNA molecules
to target and degrade specific mRNA transcripts.
• Antisense Oligonucleotides:
• Synthetic strands of DNA or RNA that bind to mRNA, preventing 448
Gene Therapy in the Treatment of Diseases
Gene therapy has shown promise in treating a variety of
diseases, including:
• Genetic disorders:
• Cystic fibrosis, sickle cell anemia, hemophilia
• Cancer:
• Gene therapy can be used to introduce tumor-suppressor genes
or to target and destroy cancer cells.
• Infectious diseases:
• Gene therapy can be used to confer resistance to infections or to
enhance the immune response.
• Neurological disorders:
• Gene therapy has shown potential for treating diseases such as
Parkinson's disease and Alzheimer's disease.
• Viral Infections:
• Therapeutic genes can be used to enhance the immune response
or inhibit viral replication, as seen in HIV research.
• Cardiovascular Diseases:
• Gene therapy can be used to promote angiogenesis or protect
against ischemic damage. 449
Challenges and Future of Gene Therapy
• Despite its potential, gene therapy faces several challenges:
• Delivery:
• Efficient and safe delivery of therapeutic genes to target
cells remains a significant hurdle.
• i.e. Efficiently delivering genes to the right cells
remains a significant hurdle.
• Immune response:
• The body's immune system can recognize and attack
foreign DNA, limiting the effectiveness of gene therapy.
• Long-term effects:
• The long-term safety and efficacy of gene therapy need
to be carefully evaluated.
• Regulation and Ethics:
• Regulatory frameworks for gene editing and therapy are
still developing, raising ethical concerns.
• Cost:
• The high cost of developing and administering gene
therapy treatments limits accessibility. 450
Future Directions
• Innovative Delivery Systems:
• Advances in nanotechnology and biomaterials may
improve gene delivery.
• Personalized Medicine:
• Tailoring gene therapies to individual genetic profiles
could enhance efficacy.
• Combination Therapies:
• Combining gene therapy with other treatments, such as
immunotherapy, may improve outcomes.
• Long-term Efficacy Studies:
• Ongoing research to understand long-term effects and
safety profiles of gene therapies.

• However, ongoing research and technological

advancements are addressing these challenges. The
future of gene therapy holds promise for treating a
wide range of diseases and improving human health. 451
• Automated DNA sequencing
In 1986, Leroy Hood and Lloyd Smith automated Sanger’s method. In this new sequencing
technology,
radioactive markers are replaced with fluorescent ones. Each ddNTP terminator is tagged with
a different
color of fluorophore: red, green, blue, or yellow. Thus, instead of having to run four separate
sequencing
reactions, the reactions can be combined into one tube. The first automated sequencer made
use of a
polyacrylamide gel to resolve the samples, a laser to excite the dye molecules as they reached
a detector near
the end of the gel, and a computer to read the results as a DNA sequence. In this system each
automated
sequencer was able to produce 4800 bases of sequence per day. The current automated
systems replace the
old-style gel with arrays of tiny capilliaries, each of which acts as a “lane.” A pump loads
special capillaries
with a polymer that serves as the separation matrix. DNA samples in a 96-well plate are loaded
into the
capillary array by a short burst of electrophoresis, called “electrokinetic injection.” The
capillary array is
immersed in running buffer and the DNA fragments then migrate through the capillary matrix
by size,
smallest to largest. As the DNA fragments reach the detection window, a laser beam excites
the dye
molecules causing them to fluoresce. Emitted light from 96 capillaries is collected at once,
spectrally
separated into the four colors and focused onto a CCD camera. Computer software interprets
the pattern of
peaks to produce a graph of fluorescence intensity versus time (electropherogram), which is
then converted
to the DNA sequence (Fig. 8.16). With this system, as many as 2 million bases can be
sequenced per day
452
453
The End

Thank You