Recombinant DNA Technology

Recombinant DNA
Technology
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”
It involves the application of scientific
principles to the processing of
materials by biological agents.
 Genetic recombination is the
exchange of information between
two DNA segments.
 This is a common occurrence within the
same species.
 But by artificial means, when a gene of one
species in transferred to another living
organism, it is called recombinant DNA
technology.
 In common, this is known as genetic
engineering.
Definition of
recombinant DNA
 Production of a unique DNA
molecule by joining together two or
more DNA fragments not normally
associated with each other
 DNA fragments are usually derived

from different biological sources
Definition of recombinant
DNA technology
 A series of procedures used to

recombine DNA segments. Under
certain conditions, a recombinant DNA
molecule can enter a cell and
replicate.
History of recombinant
DNA technology
 Recombinant DNA technology is
one of the recent advances in
biotechnology, which was
developed by two scientists
named Boyer and Cohen in
1973.
Development of
molecular biology
 Early research on prokaryotic genetics and
the development of molecular techniques
has led to a new discipline called
MOLECULAR BIOLOGY
 “Tools” have been developed (and still
continue to be modified/improved) to enable
scientists to examine very specific regions
of the genome or genes.
• Advances in Molecular Biology
– The combination of
restriction/modification enzymes
and hybridization techniques
enable the application of a wide
variety of procedures
TECHNIQUES and PROCEDURES
 Gene isolation/purification/synthesis
 Sequencing/Genomics/Proteomics
 Polymerase chain reaction (PCR)
 Mutagenesis (reverse genetics)
 Expression analyses (transcriptional and
translational levels)
 Restriction fragment length polymorphisms
(RFLPs)
 Biochemistry/ Molecular modeling
 Gene therapy
Applications
 Quantitative preparation of
biomolecules
 Recombinant Vaccines
 Antenatal diagnosis of genetic
diseases
 Monoclonal antibodies
 Cell/tissue culture
 To identify mutations in genes
 Xenotransplantation
Contd…
 To detect activation of
oncogenes
 Production of next
generation antibiotics
 Forensics
 Biosensors
 Genetically modified crops
 Bioterrorism detection
1.Quantitative Preparation of
Biomolecules
 If molecules are isolated from higher

organisms, the availability will be
greatly limited.
 For eg.- To get 1 unit of growth
hormone, more than 1000 pituitaries
from cadavers are required.
 By means of recombinant technology,
large scale availability is now assured.
2. Risk of contamination is Eliminated
• It is now possible to produce a

biological substance without any
contamination.
• Hepatitis, caused by HBV, is highly
contagious.
• Preparations of vaccines or clotting factors
are free from contaminants such as hepatitis
B particles.
• RD-Technology provides the answer to
produce safe antigens for vaccine production.
3. Specific probes for Diagnosis of
Diseases
Specific probes are useful for:

i. Antenatal diagnosis of genetic diseases.
For eg.- many of the single gene
defects like cystic fibrosis, phenyl
ketonuria etc.
Could be identified by taking cell
samples from fetus.
ii. To identify viral particles or bacterial DNA
in suspected blood and tissue samples.
Contd…
iii. To demonstrate virus

integration in transformed cells.
iv. To detect activation of
oncogenes in cancer.
v. To pinpoint the location of a gene
in a chromosome.
vi. To identify mutations in genes.
4. Gene Therapy
It is an important applications

of RD-Technology
Normal genes could be
introduced into the patient so
that genetic diseases can be
cured.
• How to find one gene in large genome?
• A gene might be 1/1,000,000 of the genome.
Three basic approaches:
• 1. Cell-based molecular cloning: create and
isolate a bacterial strain that replicates a copy of
your gene.
• 2. Polymerase chain reaction (PCR).
Make many copies of a specific region of
the DNA.
• 3. Hybridization: make DNA single stranded,

allow double strands to re-form using a labeled
(e.g. radioactive) version of your gene to make it
easy to detect.
Basic principle of recombinant
DNA technology
 The DNA is inserted into another

DNA molecule called ‘vector’
 The vector is then

introduced
recombinantinto a host where
replicates cell the itis then
itself, produced gene
Cell-Based Molecular
Cloning
• The original recombinant DNA technique: 1974
by Cohen and Boyer.
•
• Several key players:
• 1. restriction enzymes. Cut DNA at specific
sequences. e.g. EcoR1 cuts at GAATTC and BamH1
cuts at GGATCC.
•
– Used by bacteria to destroy invading DNA: their
own DNA has been modified (methylated) at the
corresponding sequences by a methylase.
•
2.Plasmids: independently replicating DNA circles (only
circles replicate in bacteria). Foreign DNA can be
inserted into a plasmid and replicated.
– Plasmids for cloning carry drug resistance genes

that are used for selection.
– Spread antibiotic resistance genes between bacterial
species
3. DNA ligase. Attaches 2 pieces of DNA together.
4.transformation: DNA manipulated in vitro can be put

back into the living cells by a simple process .
– The transformed DNA replicates and expresses its
genes.
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II.Restriction
Endonucleases
A. Origin and
function
• Bacterial origin = enzymes that cleave foreign
DNA
• Named after the organism from which they
were derived
– EcoRI from Escherichia coli
– BamHI from Bacillus amyloliquefaciens
• Protect bacteria from bacteriophage
infection
– Restricts viral replication
• Bacterium protects it’s own DNA by
methylating those specific sequence.
B. Availability
• Over 200 enzymes identified, many

available commercially from
biotechnology companies
C.
Classes
 Type I
– Cuts the DNA on both strands but at a non-
specific location at varying distances from
the particular sequence that is recognized
by the restriction enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications
• Type II
– Cuts both strands of DNA within the
particular sequence recognized by the
restriction enzyme
– Used widely for molecular biology
procedures
– DNA sequence = symmetrical
• Reads the same in the 5’- 3’ direction on
both strands = Palindromic Sequence
• Some enzymes generate “blunt ends” (cut in
middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments
together by forming phosphodiester bonds of the
phosphate-sugar backbones
DNA Ligase in Action!

Recombinant DNA Technology

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Recombinant DNA

 DNA fragments are usually derived

 A series of procedures used to

 If molecules are isolated from higher

• It is now possible to produce a

Specific probes are useful for:

iii. To demonstrate virus

It is an important applications

• 3. Hybridization: make DNA single stranded,

 The DNA is inserted into another

 The vector is then

– Plasmids for cloning carry drug resistance genes

4.transformation: DNA manipulated in vitro can be put

• Over 200 enzymes identified, many

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