RECOMBINANT DNA

TECHNOLOGY
What is Recombinant DNA technology???
Set of experimental methods concerned with
piecing together DNA segments of diverse origin,
creating sequences that would not otherwise be
found in the genome and inserting into a host
organism to produce new genetic combinations
that are of value to science, medicine, agriculture,
and industry.

Recombinant DNA is the general name given to the

piece of DNA that has been created by the
combination of at least two strands. Recombinant
DNA molecules are also called chimeric DNA
 Why recombinant DNA is produced???
Since the focus is on the gene, the fundamental goal is to
isolate, characterize, and manipulate genes:

• To obtain a large number of copies of specific DNA

segments.

• To recover large quantities of the protein produced by

concerned gene.

• To integrate the gene in question into the chromosome

of target organism where it expresses itself.
 HISTORY

The idea of recombinant DNA was first proposed by

Peter Lobban, in the Biochemistry Department at
Stanford University Medical School. First
Recombinant DNA was achieved in 1973 by Herbert
W. Boyer, of the University of California at San
Francisco, and Stanley N. Cohen, at Stanford
University. The first licensed drug generated using
recombinant DNA technology was human insulin,
developed by Gentech.
 Steps involved in RDT
1. Preparation of DNA to be
cloned.
2. Insertion of the isolated gene in
a suitable vector to obtain
recombinant DNA.
3. Introduction of the recombinant
DNA into suitable organism
called host (transformation).
4. Selection of the transformed
host cells, and identification of
the clone containing the desired
gene.
5. Multiplication/Expression of the
introduced gene in the host.
 1. Preparation of DNA to be cloned
• The genomic DNA free of
proteins and RNA is
extracted from the organism
of interest.

• DNA may also be obtained

from RNA (cDNA), or in the
form of synthetic DNA.
• Polymerase chain reaction is used for amplification of specific
DNA sequences prior to molecular cloning.

• The DNA is then treated with a restriction enzyme to generate

fragments with ends capable of being linked to those of the
vector.
 Restriction enzymes
A restriction enzyme is an enzyme that cleaves DNA into
fragments at or near specific recognition sites within the DNA
molecule known as restriction sites. They produce a double-
stranded cut in the DNA

Cohesive ends

Blunt ends
Some restriction enzymes, their origin
and recognition sequence
 2. Insertion of the isolated gene in a
suitable vector to obtain recombinant DNA.
• The cloning vector is treated
with a restriction enzyme to
cleave it at the site where
foreign DNA will be inserted.
• The vector DNA and foreign
DNA are cut with the same
restriction enzyme.
• Recombinant DNA is
obtained by mixing together
the cleaved vector and
foreign DNA and exposing to
DNA ligase that covalently
links the ends together
 Vector
A vector is a DNA molecule that has the ability to replicate
autonomously in an appropriate host cell and into which the DNA
fragment to be cloned is integrated.

Characteristics of a Cloning Vector:

• Origin of replication (ORI): specific sequence of nucleotide in DNA
from where replication starts. This process marks autonomous
replication in vector.
• Unique restriction sites to facilitate cloning of insert DNA
• Selectable marker gene: it allows the selection of the host cells,
which carry the recombinant DNA.
• Multiple cloning sites
• Small in size: large DNA molecules are broken during purification
procedure.
 Types of cloning vectors
Cloning vectors: Vectors used for propagation of DNA inserts in a
suitable host.
Expression vectors: Vectors designed for the production of the
protein specified by the DNA insert. Such vectors contain regulatory
sequences such as, promoters, operators, ribosomal binding sites
etc.
Types of vectors Size of DNA that
can be inserted
Plasmid (A DNA molecule other than bacterial chromosome) 0.5 to 8 kb
Bacteriophage (Viruses that attack bacteria) 9 to 25 kb
Cosmid (Plasmids having certain features of λ phage ) 30 to 45 kb
Artificial chromosomes (Circular or linear vectors that are stably maintained in 1 or 2
copy per cell)
Yeast Artificial Chromosome (YAC) 250 to 1000 kb
Bacterial Artificial Chromosome (BAC) 50 to 300 kb
pUC vector
Characteristics of pUC vector:

• N-terminal fragment of β-
galactosidase (lacZ) gene
of E.coli.
• The multiple cloning
site (MCS) region in the lacZ
gene, providing
many restriction sites.
• Ampicillin resistance gene
(ampR)
• Origin of replication.
 3. Introduction of the recombinant DNA
into suitable organism called host
Competence of Bacteria
Not all bacteria are capable of taking up exogenous
DNA from their environment. The practical approach
to acquire competence is to make the bacterial cells
artificially competent using:
• Chemicals containing bivalent cations (most
commonly CaCl2) and recombinant vectors are
then introduced in bacterial cells by co-incubation
and a subsequent heat shock step.
• Electrical pulses: Plasmids can also be introduced
into the electro competent host cell via
electroporation. During this procedure, cells co-
incubated with plasmids are exposed to short
pulses of electric shock.
 4. Selection and multiplication of the
transformed host cells
Several methods are employed for selection of transformed cells:
• Antibiotic resistance
• Visible characters
• Assay for biological activity
• Colony hybridization
• Blotting test
 Blue white colony selection
The host E. coli strain carries the lacZ deletion mutant (lacZΔM15) which contains the ω-
peptide, while the plasmids used carry the lacZα sequence which encodes the first 59
residues of β-galactosidase, the α-peptide. Neither is functional by itself. However, when
the two peptides are expressed together, as when a plasmid containing
the lacZα sequence is transformed into a lacZΔM15 cells, they form a functional β-
galactosidase enzyme which gives blue color in the presence of X-Gal (5-bromo-4-chloro-
3-indolyl-β-D-galactopyranoside) and IPTG (Isopropyl β-D-1-thiogalactopyranoside)
 Applications of RDT
Applications in Crop Improvement:

• Development of Transgenic Plants: Resistance to diseases,

insects and pests, herbicides, drought; metal toxicity
tolerance; induction of male sterility for plant breeding
purpose; and improvement of quality can be achieved through
recombinant DNA technology.
• Nitrogen Fixation: The Rhizobium genes responsible for
nitrogen fixation can now be transferred to cereal crops like
wheat, rice, maize, barley etc. through the techniques of
genetic engineering thus making these crops too capable of
fixing atmospheric nitrogen.
• Grains with Improved Nutritional Characteristics: For
example, Golden rice that makes α- carotene, which is
converted into vitamin A by humans.
 Continued….
Applications in Medicines:
• Production of Antibiotics: Penicillium and Streptomyces fungi are
used for mass production of famous antibiotics penicillin and
streptomycin.
• Production of Vaccines: Vaccines are now produced by transfer of
antigen coding genes to disease causing bacteria. Such antibodies
provide protection against the infection by the same bacteria or virus.
• Production of Enzymes: Some useful enzymes can also be produced
by recombinant DNA technique. For instance, enzyme urikinase,
which is used to dissolve blood clots, has been produced by
genetically engineered microorganisms.
• Gene Therapy: In gene therapy a genetic disorder is treated by
introducing gene of the lost functional protein into the patient’s cells.
• Diagnosis of Disease: Recombinant DNA technology has provided a
broad range of tools to help physicians in the diagnosis of diseases.
 Continued….
• Industrial application: Recombinant DNA technique help in
the production of chemical compounds of commercial
importance, improvement of existing fermentation processes
and production of proteins from wastes.

• Solution of Disputed Parentage: Disputed cases of parentage

can now be solved most accurately by recombinant technology
than by blood tests.

• Keeping Our Environment Pollution Free: A vast majority of

applications of environmental biotechnology use naturally
occurring micro-organisms (bacteria, fungi, etc.) to identify
and filter manufacturing waste before it is introduced into the
environment.
 Bt Cotton
Bt cotton is a genetically modified pest resistant cotton plant, which produces an
insecticide to bollworm. It is the first transgenic crop plant to be introduced in India in
2002 by a joint venture between Monsanto and Mahyco. Bt cotton has been genetically
modified by the insertion of one or more genes of insecticidal proteins from a common
soil bacterium, Bacillus thuringiensis which produce over 200 different Bt toxins, each
harmful to different insects. Thus, genetically transformed plants produce one or more
toxins as they grow. The genes that have been inserted into cotton produce toxins that are
limited in activity almost exclusively to caterpillar pests (Lepidoptera or bollworm).
 Concerns related to GMOs
Concerns regarding genetically engineered microorganisms:
• They may rapidly multiply and out-compete the native
microbes.
• They may transfer genes related to virulence into the native
bacterial populations and thereby increase their virulence.
Concerns regarding transgenic plants:
• Production of toxic or allergic metabolites for consumers
• Unexpected new suceptibilities to pathogens
• Virus resistance genes may provide oppurtunities for
evolution of newer virulent strains.
• Transmission of new traits to related sexually compatible
weed species.
• The ecosystems may be disturbed by dispersal, persistence
or altered reaction to parasites, symbionts or competitors.
 Biosafety regulations in India
India has a well-defined regulatory mechanism for development and
evaluation of GMOs including transgenic crops and the products
thereof. The two main agencies identified for implementation of the
rules are the Ministry of Environment & Forests (MoEF) and the
Department of Biotechnology (DBT), Government of India. There are
six competent authorities under MoEF and DBT:

• Recombinant DNA Advisory Committee (RDAC)

• Review Committee on Genetic Manipulation (RCGM)
• Genetic Engineering Approval Committee (GEAC)
• Institutional Biosafety Committees (IBSC) attached to every
organization engaged in rDNA research
• State Biosafety Coordination Committees (SBCC)
• District Level Committees (DLC)
 Regulatory framework in India

The regulatory framework for GMOs in India

consists of the following rules and guidelines.
Rules and policies
• Rules 1989 under Environment Protection Act
(1986)
• Seed Policy 2002
Guidelines
• Recombinant DNA guidelines, 1990
• Guidelines for research in transgenic crops, 1998
Thank You