Recombinant DNA Technology

BSc. MLT Third year

Presented by: Kailash Acharya

Presented to : Rajesh Kumar Thakur
Introduction:
• Biotechnology may be defined as "the method by which a living
organism or its parts are used to change or to incorporate a particular
character to another living organism".
• But by artificial means, when a gene of one species is transferred to
another living organism, it is called recombinant DNA technology.
• In common practice, this is known as genetic engineering.
• Genes or sets of gene can also be recombined in vitro to produce new
combinations that do not occur biologically. Recombinant DNA
technology involves isolation and manipulation of DNA to make
chimeric or hybrid DNA molecule.
• Chimeric DNA is a recombinant DNA containing genes from two
different species, e.g. molecules containing both human and bacterial
DNA sequences.
• Recombinant DNA is a DNA formed by the hybrid combination of two
DNA fragments, derived from different sources.
• The normal biological exchange or addition of genes from different
sources to form an altered chromosome, which can be replicated,
transcribed and translated, is called genetic recombination.
Basic Principle of r-DNA Technology:

• There are many diverse and complex techniques involved in gene manipulation.
However, the basic principles of recombinant DNA technology are reasonably
simple, and broadly involve the following stages;

1. Generation of DNA fragments and selection of the desired piece of DNA (e.g.
a human gene).
2. Insertion of the selected DNA into a cloning vector (e.g. a plasmid) to create a
recombinant DNA or chimeric DNA.
3. Introduction of the recombinant vectors into host cells (e.g. bacteria).
4. Multiplication and selection of clones containing the recombinant molecules.
5. Expression of the gene to produce the desired product.
1. Isolation of Specific Human DNA:

• The genomic DNA is very large in size. Isolation of a specific fragment of DNA can be
achieved by splicing or cleaving brought about by a group of key enzymes called
restriction endonucleases.
• Once DNA has been cleaved into fragments by restriction endonuclease, a particular
fragment of interest can be separated by others, by electrophoresis or HPLC.
Molecular tools for genetic engineering:
1. Restriction endonucleases:(DNA cutting enzymes)
• One of the major obstacles to molecular analysis of genomic DNA is the
immense size of the molecules involved.
• The discovery of a special group of bacterial enzymes, called restriction
endonucleases , which cleave double-stranded (ds) DNA into smaller, more
manageable fragments, opened the way for DNA analysis.
• These are the bacterial enzymes that can cut/split DNA at specific sites.
• They were first discovered in E.coli restricting the replication of
bacteriophages, by cutting the viral DNA. The host (E.coli) DNA is protected
from cleavage by addition of methyl groups.
• Thus, the enzymes that restrict the viral replication are known as restriction
enzymes or restriction endonucleases.
Specificity of restriction enzymes:
• Restriction endonucleases recognize short stretches of dsDNA (four to eight bp)
that contain specific nucleotide sequences.
• These sequences, which differ for each restriction enzyme, are palindromes, that
is, they exhibit twofold rotational symmetry.

Nomenclature:
• A restriction enzyme is named according to the organism from which it was
isolated.
• The first letter of the name is from the genus of the bacterium. The next two
letters are from the name of the species. An additional letter indicates the type or
strain, and a number (Roman numeral) is appended to indicate the order in
which the enzyme was discovered in that particular organism.
• For example, HaeIII is the third restriction endonuclease isolated from the
bacterium Haemophilus aegyptius.
Cleavage pattern:
• Majority of restriction endonucleases cut DNA at defined sites within
recognition sequence.
• Some restricted endonucleases cleave both strands of DNA, so as to
leave no unpaired bases on either end. These ends are often called
blunt ends.
• Some restricted endonucleases make staggered cuts on the two
strands, leaving two to four nucleotides of one strand unpaired at each
resulting end. These are referred to as cohesive ends or sticky ends.
• Different restriction fragments of DNA can base-pair with each other
if they have sticky ends which are complementary.
• A selected list of enzymes, recognition sequences, and their products
formed is given in table below;
2. DNA Ligases:(DNA joining enzymes)

• The cut DNA fragments are covalently joined together by DNA

ligases. These enzymes were originally isolated from viruses. DNA
ligases actively participate in cellular DNA repair process.
• The action of DNA ligases is absolutely required to permanently hold
DNA pieces. This is so since the hydrogen bonds formed between the
complementary bases of DNA strand are not strong enough to hold the
strands together.
• DNA ligase joins the DNA fragments by forming a phosphodiester
bond between the phosphate group of 5’-carbon of one deoxyribose
with the hydroxyl group of 3’-carbon of another deoxyribose .
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).
• In general, microorganisms are preferred
as host cells, since they multiply faster
compared to cells of higher organisms
(plants or animals).
2. Chimeric or Hybrid DNA:
• The main aim of genetic engineering is to insert a DNA of interest
(foreign DNA) into a vector DNA so that the DNA fragment replicates
along with the vector after annealing.
• This hybrid combination of two fragments of DNA is referred to as
chimeric DNA or hybrid DNA or recombinant DNA.

Steps of preparation:
• A circular plasmid vector DNA is first cut with a specific restriction
endonuclease. If Eco R I is used, sticky ends with TTAA sequence on one
DNA strand and AATT sequence on the other strand are produced.
• The human DNA (foreign or insert DNA) is also cut with the same
restriction endonuclease, so that the same sequences are produced on the
sticky ends of the cut piece.
 • Next, the vector DNA and human cut piece DNA are incubated together so
that annealing occurs. The sticky ends of both vector and human DNA have
complementary sequences, hence they come into contact with each other.
• The enzyme DNA Ligase is allowed to act on the hybrid or chimeric DNA
molecule. The enzyme joins the two fragments by covalent phosphodiester
linkages between the vector and insert molecules and finally the chimeric
DNA molecule is produced

Problems in preparation of chimeric DNA:

Although sticky ends ligation is technically easy, but sometimes problems may come up as
follows and some special techniques are required to overcome these:
a. Sticky ends of a vector may recombine with themselves without taking up the “insert”
molecule.
b. Sticky ends of insert fragments similarly also can anneal without joining the vector.
c. Lastly, sometimes sticky end sites may not be available.
Homopolymer Tailing:
• It is a technique by which sticky ends can be produced on a blunt
ended DNA molecule,
• If poly d (G) is added to the 3' ends of the vector and poly d (C) is
added to the 3' ends of the insert foreign DNA molecule, the two
molecules can only anneal to each other circumventing the problems
mentioned above.
• This procedure is called homopolymer tailing.

Other methods:
• Use of synthetic Linker DNA,
• Use of DNA ligase,
Vectors:(The cloning Vehicles)

• A vector is a molecule of DNA to which the fragment of DNA to be cloned is

joined.
• Essential properties of a vector include:
1) It must be capable of autonomous replication within a host cell,
2) It must contain at least one specific nucleotide sequence recognized by a
restriction endonuclease,
3) it must carry at least one gene that confers the ability to select for the vector
such as an antibiotic resistance gene.
• Commonly used vectors include plasmids, bacteriophages, cosmids and artificial
chromosome vectors.
Plasmid:
• Plasmids are extra-chromosomal, double stranded, circular, self-replicating
DNA molecules.
• Almost all the bacteria have plasmids containing a low copy number (1-4 per cell)
or a high copy number (10-100 per cell).
• The size of the plasmids varies from 1 to 500 kb.

Nomenclature of plasmids:
• It is a common practice to designate plasmid by a lower case p, followed by the first
letter(s) of researcher(s) names and the numerical number given by the workers.
• Thus, pBR322 is a plasmid discovered by Bolivar and Rodriguez who designated it
as 322.
• Some plasmids are given names of the places where they are discovered e.g. pUC is
plasmid from University of California.
pBR322 – the most common plasmid vector :
• pBR322 of E.coli is the most popular and widely used plasmid vector, and is
appropriately regarded as the parent or grand parent of several other vectors.
• pBR322 has a DNA sequence of 4,361 bp.
• It carries genes resistance for ampicillin (Ampr) and tetracycline (Tetr) that serve
as markers for the identification of clones carrying plasmids.

Other plasmid cloning vectors

• The other plasmids employed as cloning vectors include pUC19 (2,686 bp, with
ampicillin resistance gene), and derivatives of pBR322–pBR325,pBR328 and pBR329.
Fig. plasmid cloning vector pBR322
Bacteriophages:
• Bacteriophages or simply phages are the viruses that replicate within the
bacteria.
• In case of certain phages, their DNA gets incorporated into the bacterial
chromosome and remains there permanently.
• Phage vectors can accept short fragments of foreign DNA into their genomes.
• 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.
• The most commonly used phages are bacteriophage-gamma and bacteriophage
(phage M13).
Cosmids:
• Cosmids are the vectors possessing the characteristics of both plasmid and
bacteriophage-gamma.
• The advantage with cosmids is that they can carry larger fragments of foreign DNA
compared to plasmids.
Artificial chromosome vectors:
• Human artificial chromosome (HAC) :
Human artificial chromosome is a synthetically produced vector DNA, possessing
the characteristics of human chromosome.
• Yeast artificial chromosomes (YACs) :
Yartificial chromosome (YAC) is a synthetic DNA that can accept large fragments
of foreign DNA (particularly human DNA). It is thus possible to clone large DNA
pieces by using YAC.
• Bacterial artificial chromosomes (BACs) :
The construction of BACs is based on one F-plasmid which is larger than the other
plasmids used as cloning vectors. BACs can accept DNA inserts of around 300 kb.
3. Cloning of chimeric DNA:
• A clone is a large population of identical molecules, bacteria or cells that arise from a
common ancestor.
• This cloning allows for the production of a large number of identical DNA molecules which
can then be characterized or used for various purposes.

4. Transfection of vector into the host:

• The chimeric DNA contained in a plasmid vector or phages or cosmids can be introduced
into bacterial cells E. coli strain C 101 by a process called transfection.
• The process by which plasmid is introduced into the host is called transfection.
• The replicating bacterial cell (host cell) permits the amplification of the chimeric DNA of
the vector.
• In this way, cloning results in the production of large number of identical target DNA
molecules. The cloned target DNA is released from its vector by cleavage using
appropriate restriction endonucleases, isolated, characterized and used for various
purposes.
5. Plasmid carries antibiotic resistant genes:

• Plasmid pBR-325 vector contains Apr (ampicillin resistance), Tcr

(tetracycline resistance) and Cmr (chloramphenicol resistance) genes.
• The restriction enzyme EcoRI will cleave the plasmid in the middle of
Cmr gene.
• When the foreign DNA is inserted, the resistance against chloramphenicol
is lost.
• This insertional inactivation of Cmr gene is the marker for hybrid DNA.
6. Selection of Colony having Desired gene:

I. After the transfection, the bacteria are cultured in a medium containing

ampicillin and tetracycline. The antibiotics kill all the wild bacteria. Only
the bacteria containing the plasmids will grow.
II. There will be many colonies, where the vector does not carry the foreign
DNA. These colonies are replica-plated onto another agar plate
containing chloramphenicol. Since the insertion of foreign DNA
abolishes chloramphenicol resistance, the desired colonies are killed in
the replica-plate.
III. Colonies in the original plate, corresponding to the dead colonies in the
replica-plate are selected. They carry the foreign gene
IV. The selected colonies are further cultured to produce clones.
7. Expression Vectors:
• To produce the human proteins, E. coli carrying the vector with the
insert is allowed to grow, without any protein inhibitors. Such a vector
carrying the foreign gene, which is translated into a protein, is called
expression vector.
• The human proteins can be harvested from the bacterial culture.
Applications of genetic engineering:
• Molecular basis of disease: Recombinant DNA technology is used to understand the
molecular basis of a number of diseases, for example:
– Familial hypercholesterolemia
– Sickle cell disease
– Thalassemias
– Cystic fibrosis
– Muscular dystrophy, etc.
• Diagnosis of disease: Recombinant DNA technology is used to diagnose existing diseases.
• Production of proteins: Using recombinant technology, human proteins can be produced
in abundance for therapy, research and diagnosis, for example:
– Anticoagulant: Tissue plasminogen activator (TPA). Used in treating heart attack
victims.
– Human growth hormone: It is used to treat children with growth hormone deficiencies
(dwarfism)
 – Erythropoietin: Used to treat anemia.
– Blood factors: Factor VIII, used to treat hemophilic patients.
– Insulin: A hormone used to treat diabetes.
– Interferons: Used to treat cancer.
– Interleukins: Used in wound healing, HIV infections, cancer, immune deficiencies.
– Monoclonal antibodies: Used in diagnostic tests.
– Superoxide dismutase: Used during surgery.
– Vaccines: Various vaccincs can be produced by recombinant DNA technology. The
first successful recombinant DNA vaccine produced was for the hepatitis B virus.
• Gene therapy:
Gene therapy is the introduction of normal genes into individuals who have defective
genes. Gene therapy for sickle cell disease, thalassemia, adenosine deaminase deficiency
and other diseases may be devised. Currently gene therapy is at experimental level.
• Transgenic animals
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