RECOMBINANT DNA TECHNOLOGY

Recombinant DNA refers to the creation of new combinations of DNA segments that are
not found together in nature. This technique involves cutting of DNA by Restriction
endonuclease, joining it with vector i.e preparing a chimeric molecule, inserting it to host
and cloning the molecule. The isolation and manipulation of genes allows for more
precise genetic analysis as well as practical applications in medicine, agriculture, and
industry.

TOOLS FOR RDT

A] Enzymes

• Restriction endonucleases- fragment the DNA into defined segments

• DNA Ligase- joins DNA fragments
• DNA polymerase-Synthesizes DNA based on a template; Fills in gaps in DNA
• Reverse transcriptase- Copies RNA into DNA “cDNA”
• Ribonucleases (endo and exo)
• RNA polymerase
• Terminal transferase- For radiolabeling
• Polynucleotide kinase- For radiolabeling
• Alkaline phosphatase- To remove phosphate; 3’ or 5

B] complementary DNA

(cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by

the enzyme reverse transcriptase and the enzyme DNA polymerase.

Synthetic DNA: Produced by chemical process

Random DNA: Cut by RE.

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-
stranded or single stranded DNA at specific recognition nucleotide sequences known
as restriction sites. Restriction enzymes recognize a specific sequence of
nucleotides and produce a double-stranded cut in the DNA. While recognition
sequences vary between 4 and 8 nucleotides, many of them are palindromic, which
correspond to nitrogenous base sequences that read the same backwards and
forwards. EcoRI digestion produces "sticky" ends

whereas SmaI or many other restriction enzyme cleavage produces "blunt" ends

Recognition sequences in DNA differ for each restriction enzyme, producing differences
in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-
end "overhang" of an enzyme restriction.

Restriction endonucleases are categorized into four general groups

 Type I enzymes cleave at sites remote from recognition site; require both ATP
and SAM to function
 Type II enzymes cleave within or at short specific distances from recognition site;
most require magnesium
 Type III enzymes cleave at sites a short distance from recognition site; require
ATP (but doesn't hydrolyse it)
 Type IV enzymes target methylated DNA.
Examples of restriction enzymes include:

Recognition
Enzyme Source Cut
Sequence

5'GAATTC 5'---G AATTC---3'

EcoRI Escherichia coli
3'CTTAAG 3'---CTTAA G---5'

5'CCWGG 5'--- CCWGG---3'

EcoRII Escherichia coli
3'GGWCC 3'---GGWCC ---5'

5'---G GATCC---
Bacillus 5'GGATCC 3'
BamHI
amyloliquefaciens 3'CCTAGG 3'---CCTAG G---
5'

5'AAGCTT 5'---A AGCTT---3'

HindIII Haemophilus influenzae
3'TTCGAA 3'---TTCGA A---5'

Vectors

a cloning vector

• Should be capable of replicating in host cell

• Should have convenient RE sites for inserting DNA of interest
• Should have a selectable marker to indicate which host cells received
recombinant DNA molecule
• Should be small and easy to isolate
Plasmid: Bacterial plasmids are small, circular, extra chromosomal DNA molecules that
are separate from the rest of the chromosome. They replicate independently of the
bacterial chromosome. Useful for cloning DNA inserts less that 10 kb (kilobase pairs).

pBR 322

Bacteriophage lambda (45 kb) contains a central region of 15 kb that is not required
for replication or formation of progeny phage in E. coli. Thus, lambda can be used as a
cloning vector by replacing the central 15 kb with 10-15 kb of foreign DNA.

Cosmids are hybrids of phages and plasmids that can carry DNA fragments upto 45 kb.
They can replicate like plasmids but can be packaged like phagelambda. Expression
vectors are vectors that carry host signals that facilitate the transcription and translation
of an inserted gene. They are very useful for expressing eukaryotic genes in bacteria.

Yeast artificial chromosomes (YACS) are yeast vectors that have been engineered to
contain a centromere, telomere, origin of replication, and a selectable marker. They
can carry up to 1,000 kb of DNA. Since they are maintained in yeast (a eukaryote), they
are useful for cloning eukaryotic genes that contain introns. Also, eukaryotic genes are
more easily expressed in a eukaryotic host such as yeast.
Bacterial artificial chromosomes (BACS) are bacterial plasmids derived from the F
plasmid. They are capable of carrying up to 300 kb of DNA

STEPS OF RDT

Step 1: Isolation of vector and gene-source DNA

• Bacterial plasmids and foreign DNA containing the gene of interest are
isolated
• plasmid is from E. coli (pBR322) and has two genes:
ampR which confers antibiotic resistance to ampicillin and Ter gene for
tetracycline resistance
Step 2: Insertion of gene-source DNA into the vector

(a) Digestion
 • The restriction enzyme cuts plasmid DNA at the restriction site
• The foreign DNA is cut into thousands of fragments by the same
restriction enzyme; one of the fragments contains the gene of interest.
• When the restriction enzyme cuts, it produces sticky ends on both the
foreign DNA fragments and the plasmid

(b) Mixture of foreign DNA fragments with clipped plasmids

• Sticky ends of the plasmid base pair with
complementary sticky ends of foreign DNA fragments.
(c) Addition of DNA ligase
• DNA ligase catalyzes the formation of covalent bonds,
joining the two DNA molecules and forming a new plasmid with
recombinant DNA
Step 3: Introduction of cloning vector into bacterial cells

• The naked DNA is added to a bacterial culture.

• Some bacteria will take up the plasmid DNA by
transformation
Step 4: Cloning of cells (and foreign DNA)

• Bacteria with the recombinant plasmid are allowed to

reproduce, cloning the inserted gene in the process
Step 5: Recombinant plasmids can be identified by the fact that they are
ampicillin resistant and will grow in the presence of ampicillin.
Step 6: The identified recombinant bacterial colony was allowed to
reproduce again in a culture medium and the gene product is purified and
isolated.

Applications of Recombinant DNA technologies

A. Construction of genetic “libraries” to assist in isolation / characterization of specific
genes and gene products

Human Genome Project (and others…..)

B. Production of proteins using expression vectors

C. Production of vaccines, hormones, blood clotting factors, etc.

D. Diagnostics: antibody and oligonucleotide probes:

• cancer cells, detection / imaging

• bacterial and viral diseases (Legionnaires, AIDS)
• Prenatal diagnoses
• Forensics
E. Therapeutics: Human insulin, Human growth hormone, interferons, growth factors,
blood clotting factors, Plasminogen activator, tumor necrosis factor, novel recombinant
antibodies, synthetic peptides as recombinant vaccines (Hep B, malaria, rabies, AIDS)

F. Gene Transfer
• Plants---increased yield, improved tolerance, herbicide res., disease res.
• Transgenic animals---production of scarce proteins (eg in cow’s milk), studies of
eukaryotic gene regulation
• Human gene therapy
G. In Agriculture:

• Disease resistant crop

• Drought resistant crops

• Increases productivity and quality of crop

 Southern blotting

It is technique used to detect specific DNA fragment from mixture of DNA fragments. A
cDNA probe of gene of interest is used in this technique. This probe hybridizes with the
gene of interest thus leading to its identification. Different steps of southern blot are
given below.
1. Chromosal DNA is purified and cleaved by restriction endo nuclease. A mixture of
DNA fragments are obtained.
2. The fragments are separated by agarose gel electrophoresis. The gel is prepared
from agarose in a suitable buffer. The DNA fragments are loaded into a well at one end
of the gel and electrophoresid. Under conditions of experiment DNA fragments carry net
negative charge and hence they move towards anode. Since the gel act as molecular
sieve movements of DNA fragments towards anode depends on their sizes. Small
fragments move faster where as big fragments get retarded. Thus, the DNA fragments
are separated according to size.
3. The DNA in the separated fragments is denatured and made single stranded by
soaking gel first in a HCl then in NaOH.
4. DNA is transferred to nitrocellulose in this step. This is carried out first by placing
nitrocellulose sheet on the gel. Next the buffer in the gel is removed with help of
several blotting papers. The blotting paper when placed on nitrocellulose sheet draws
DNA also with buffer. DNA sticks to nitrocellulose and only the buffer passes through
nitrocellulose and absorbed by blotting paper. Thus a perfect nitrocellulose print of the
gel is obtained.
5. Now nitrocellulose sheet is incubated with buffer containing cDNA probe. cDNA
probe is P32 radiolabelled.
6. cDNA probe hybridizes with gene of interest and excess probe is washed off.
7. Nitro cellulose sheet is dried and placed next to x-ray film for autoradigraphy
 GENE THERAPY

Gene therapy is the insertion, alteration, or removal of genes within an

individual's cells and biological tissues to treat disease. It is a technique for correcting
defective genes that are responsible for disease development. The most common form
of gene therapy involves the insertion of functional genes into an unspecified genomic
location in order to replace a mutated gene, but other forms involve directly correcting
the mutation or modifying normal gene that enables a viral infection. Although the
technology is still in its infancy, it has been used with some success.

Gene therapy may be classified into the two following types:

Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by
the introduction of functional genes, which are integrated into their genomes. Therefore,
the change due to therapy would be heritable and would be passed on to later
generations. This new approach, theoretically, should be highly effective in
counteracting genetic disorders and hereditary diseases. However, many jurisdictions
prohibit this for application in human beings, at least for the present, for a variety of
technical and ethical reasons.

Somatic gene therapy

In the case of somatic gene therapy, the therapeutic genes are transferred into
the somatic 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.
 PCR

The polymerase chain reaction (PCR) is a scientific technique in molecular

biology to amplify a single or a few copies of a piece of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA sequence.

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR
methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although
some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components
include:

 DNA template that contains the DNA region (target) to be amplified.

 Two primers that are complementary to the 3' (three prime) ends of each of
the sense and anti-sense strand of the DNA target.
 Taq polymerase or another DNA polymerase with a temperature optimum at
around 70 °C.
 Deoxynucleotide triphosphates (dNTPs), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.
 Buffer solution, providing a suitable chemical environment for optimum activity
and stability of the DNA polymerase.
 Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but
Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher
Mn2+concentration increases the error rate during DNA synthesis
 Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction
tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the
reaction tubes to achieve the temperatures required at each step of the reaction (see
below). Many modern thermal cyclers make use of thePeltier effect, which permits both
heating and cooling of the block holding the PCR tubes simply by reversing the electric
current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for
rapid thermal equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid
require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure
Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at
~65 °C (3) Elongation at 72 °C.

 Denaturation step: This step is the first regular cycling event and consists of
heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the
DNA template by disrupting the hydrogen bonds between complementary bases,
yielding single-stranded DNA molecules.

 Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40

seconds allowing annealing of the primers to the single-stranded DNA template.
Typically the annealing temperature is about 3-5 degrees Celsius below the Tm
(melting temp.) of the primers used. Stable DNA-DNA hydrogen bonds are only
formed when the primer sequence very closely matches the template sequence. The
polymerase binds to the primer-template hybrid and begins DNA synthesis.

 Extension/elongation step: The temperature at this step depends on the DNA

polymerase used; Taq polymerase has its optimum activitytemperature at 75–80 °C
and commonly a temperature of 72 °C is used with this enzyme. At this step the
DNA polymerase synthesizes a new DNA strand complementary to the DNA
template strand by adding dNTPs that are complementary to the template in 5' to 3'
direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl
group at the end of the nascent (extending) DNA strand. The extension time
depends both on the DNA polymerase used and on the length of the DNA fragment
to be amplified.

USES OF PCR

• For amplification of DNA

• PCR permits early diagnosis of malignant diseases such

as leukemia and lymphomas. PCR assays can be performed directly on genomic
 DNA samples to detect translocation-specific malignant cells at a sensitivity that
is at least 10,000-fold higher than that of other methods

• PCR also permits identification of non-cultivatable or slow-growing

microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue
culture assays and animal models. The basis for PCR diagnostic applications in
microbiology is the detection of infectious agents and the discrimination of non-
pathogenic from pathogenic strains by virtue of specific genes.

• Viral DNA can likewise be detected by PCR. The amount of virus ("viral load") in
a patient can also be quantified by PCR-based DNA quantitation techniques.

RFLP

In molecular biology, restriction fragment length polymorphism, or RFLP is a

technique that exploits variations in homologous DNA sequences. It refers to a
difference between samples of homologous DNA molecules that come from differing
locations of restriction enzyme sites, and to a related laboratory technique by which
these segments can be illustrated. RFLPs are visualized by digesting DNA from
different individuals with restriction endonucleases, followed by gel electrophoresis to
separate fragments according to size, then Southern blotting and hybridization to a
labeled probe that identifies the locus under investigation.
 DNA Fingerprinting

1) Isolation of DNA. DNA must be recovered from the cells or tissues of the
body. Only a small amount of tissue, like blood, hair, or skin, is needed.
For example, the amount of DNA found at the root of one hair is usually
sufficient.

2) Cutting, sizing, and sorting. Special enzymes called restriction

enzymes are used to cut the DNA at specific places. For example, an
enzyme called EcoR1, found in bacteria, will cut DNA only when the
sequence GAATTC occurs.

The DNA pieces are sorted according to size by a sieving technique

called electrophoresis. The DNA pieces are passed through a gel made
from seaweed agarose (a jelly-like product made from seaweed). This
technique is the DNA equivalent of screening sand through progressively
finer mesh screens to determine particle sizes.

3) Transfer of DNA to nylon. The distribution of DNA pieces is

transferred to a nylon sheet by placing the sheet on the gel and soaking
them overnight.

4-5) Probing. Adding radioactive or colored probes to the nylon sheet

produces a pattern called the DNA fingerprint. Each probe typically sticks
in only one or two specific places on the nylon sheet.

6) DNA fingerprint. The final DNA fingerprint is built by using several

 probes (5-10 or more) simultaneously. It resembles the bar codes used by
grocery store scanners.

Uses of DNA Fingerprints

DNA fingerprints are useful in several areas of society. They are used by professionals
in human health and the justice system.

Diagnosis of inherited disorders

DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn
babies in hospitals around the world. These disorders may include cystic fibrosis,
hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia,
and many others. Early detection of such disorders enables the medical staff to prepare
themselves and the parents for proper treatment of the child. In some programs, genetic
counselors use DNA fingerprint information to help prospective parents understand the
risk of having an affected child. In other programs, prospective parents use DNA
fingerprint information in their decisions concerning affected pregnancies.

Developing cures for inherited disorders

Research programs to locate inherited disorders on the chromosomes depend on the

information contained in DNA fingerprints. By studying the DNA fingerprints of relatives
who have a history of some particular disorder, or by comparing large groups of people
with and without the disorder, it is possible to identify DNA patterns associated with the
disease in question. This work is a necessary first step in designing an eventual genetic
cure for these disorders.
Forensic or criminal

FBI and police labs around the U.S. have begun to use DNA fingerprints to link
suspects to biological evidence-blood or semen stains, hair, or items of clothing-found
at the scene of a crime. Since 1987, more than 150 cases have been decided with the
assistance of DNA fingerprint evidence. Another important use of DNA fingerprints in
the court system is to establish paternity in custody and child support litigation. In these
applications, DNA fingerprints bring an unprecedented, nearly perfect accuracy to the
determination.

Personal identification

Because every organ or tissue of an individual contains the same DNA fingerprint, the
U.S. armed services have just begun a program to collect DNA fingerprints from all
personnel for use later, in case they are needed to identify casualties or persons
missing in action. The DNA method will be far superior to the dogtags, dental records,
and blood typing strategies currently in use.