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I M Pharm
Department of Pharmacology
• It is technique used in genetic engineering
that involves the identification, isolation and
insertion of gene of interest into a vector such
as a plasmid or bacteriophage to form a
Recombinant DNA molecule.
Steps involved in the production of
• Isolation of specific DNA
• Selection of vectors
• Preparation of rDNA
• Transfer of rDNA to host cell
• Identification and isolation of rDNA
ISOLATION OF SPECIFIC DNA
• Random fragments of source DNA can be obtained
by mechanical shearing of bacterial, plant or
• Mechanical shearing caused by high speed mixing
at 1500 rpm for 30 min. This gives fragments of
• Short single stranded regions- termini or blunt
end fragments are formed.
• Sonication can reduce the length of the fragments
to about 300 nucleotide pairs.
• Shearing does not necessarily produce 5’
phosphate and3’ ends . Therefore the end of the
fragments must be repaired.
Restriction endonuclease digestion
• Large number of restriction enzymes which recognize
and cut DNA with in target sites of nucleotides are
• Depending upon the number of target sites present,
DNA may be cut into too small or too big size
• Generally the same restriction enzyme is used for
vector and the DNA of interest.
• The digestion carried out may be light ,moderate or
heavy producing from small number to large
number of fragments which are reproducible. 6
Reverse transcriptase method
• If eukaryotic gene is to be cloned and expressed in prokaryotic
cell, the difference in gene organization has to be considered.
• The introns present in eukaryotic genes are transcribed into
mRNA. Such precursor mRNA in eukaryotic cell undergoes
post transcriptional modification and removal of introns
occurs to give rise to processed mRNA.
• Mature mRNA molecules from animal cell do not contain
sequences complementary to introns as they are removed by
• These molecules then can be directly transcribed into DNA
using an enzyme reverse transcriptase.
• cDNA thus produced for a particular protein can be joined to
appropriate vector and cloned into a host.
• Hybridization method depends on the principle that on mRNA
forms a complex with complementary DNA segments from
which it has been transcribed.
• This method is possible if protein encoding gene does not
• DNA from the donor organism is first isolated. This ds DNA is
treated with heat or alkali and is converted to ss DNA by
• Strands are then mixed with mRNA transcribed by the gene.
mRNA pairs with cDNA portion to form DNA-RNA complex
• This complex is then isolated and DNA separated from RNA.
• Ss DNA thus obtained can be converted to ds DNA by DNA
Separation of isolated DNA fragments
• The cutting of DNA by different isolation
methods, these fragments can be separated
by a technique known as gel electrophoresis.
• Electrophoresis is a separation technique, where
the compounds are separated due to varying
behavior under the influence of applied electric
• In Gel Electrophoresis, the separation is brought
about through molecular sieving technique,
based on the molecular electrophoresis by gel
method at two or more pH values. It is parallel to
charaterize the newly disovered protein
SELECTION OF VECTORS
• Vectors are used to transform DNA material
into the host cell.
• Types of vectors:
• Cloning vectors and expression vectors
• Cloning vectors: propagation of DNA inserts.
• Expression vectors: production of proteins.
• Vector is a “molecular carrier” or a “molecular
vehicle” . It is used as a mode of
transportation or transference to insert and
amplify a gene into a target genome.
• Since DNA fragments are not capable of self
replication in a host cell, a vector is used.
Selection of cloning vectors depends on ,
• Objective of cloning experiment
• Ease of working
• Knowledge existing about the vector
Properties of vectors:
• Able to self replicate in the host
• Easy to isolate
• Non toxic to host cells
• Have space for foreign inserts
• Have unique restriction sites for common
Practical features of DNA cloning vectors
• Origin of replication
• Multiple cloning sites
• Selectable marker genes
• RNA polymerase promoter sequence
• DNA sequencing primers
Plasmid cloning vectors
• Plasmids are circular ,double stranded DNA molecules that
exist in bacteria and in the nuclei of some eukaryotic cells.
• They can replicate independently of the host cell . The size
of plasmids ranges from few kb to near 100 kb
• Can hold up to 10 kb fragments
• Plasmids have an origin of replication , antibiotic resistance
genes as markers ,and several unique restriction sites.
• After culture growth the clone fragment can be recovered
easily. The cells are lysed and the DNA is isolated and
• DNA fragment can be kept indefinitely if mixed with
glycerol in a -70 degree c freezer.
Phage cloning vectors
• Fragments up to 23 kb can be accommodated by a phage vector .
• Lambda is most common phage
• 60% of the genome is needed for lytic pathway
• Segments of the lambda DNA is removed and a stuffer fragment is
• The stuffer fragment keeps the vector at a correct size and carries
marker genes that are removed when foreign DNA is inserted into
• Example: charon 4A lambda
• When charon 4A lambda is intact, beta galactosidase reacts with
xgal and the colonies turn blue.
• When the DNA segment replaces the stuffer region , the lac5 gene
is missing, which codes for beta galactosidase, no beta
galactosidase is formed and the colonies are white,
Cosmid cloning vectors:
• Fragments from 30 to 46kb can be accommodated by a
• Cosmids combine essential elements of a plasmid and
• Cosmids are extracted from bacteria and mixed with
• Cleaved cosmids are mixed with foreign DNA that has been
cleaved with the same endonucleases.
• Recombinant cosmids are packaged into lambda casp5 ds.
• Recombinant cosmid is injected into the bacterial cell
where the rcosmids arranges into a circle and replicates as
a plasmid . It can be maintained recovered just as plasmids.
Yeast artificial chromosomes (YACs)
• YACs can hold up to 500kbs
• YACs are designed to replicate as plasmids in bacteria when no
foreign DNA is present. Once a fragment is inserted , YACs are
transferred to cells , they can replicate as eukaryotic chromosomes .
• YACs contain : a yeast centromere ,two yeast telomeres, a bacterial
origin of replication and bacterial selectable markers .
• YAC plasmid – yeast chromosome
• DNA is inserted to a unique restriction site and cleaves the plasmid
with another restriction endonucleases that removes a fragment of
DNA and causes YAC to become liner . Once in the cell the rYAC
replicates as a chromosome, also replicating the foreign DNA.
Bacterial artificial chromosomes
• BACs can hold up to 300 kbs
• The F factor of E.coli is capable of handling
large segments of DNA.
• RECOMBINANT BACs are introduced into
E.coli by electroportation once in the cell the
rBAC replicates like an F factor.
Insertion of target DNA into vector
• After cutting the source DNA to generate fragment of
interest , the next task of cloning is generation of
recombinant DNA molecule by joining DNA fragment of
interest to appropriate vector. After this joining has been
done, the vector along with inserted fragment can be
transferred to suitable host .
Following methods are most commonly used for joining of
DNA to vector.
• Ligation of cohesive termini
• Blunt end ligation
• Homopolymer tailing
• Use of linker molecules
• Use of adaptor molecules
Transfer of rDNA to host cell
Recombinant DNA is allowed to enter into a
suitable host cell for expression of foreign DNA.
The recombinant vector is mainly introduced
into E.coli to select the recombinant from the
unchanged vector and to obtain many copies of
the DNA insert or recombinant vector. The
specific method is selected for transformation,
it depends on types of vectors and host cells.
ISOLATION OF RDNA
The main objective of cloning experiment is to isolate
the cells that contain recombinant vector from non
transformed cells. Recombinant cells express the
characters while the non recombinants do not express
the characters or traits .Different methods are used for
screening or selection of recombinats.
• Direct selection
Many times cloned DNA itself codes for resistance to the
antibiotic ampicillin and the recombinants can be
allowed to grow on minimal medium containing
ampicillin . Such recombinants contain ampr gene on its
plasmid vector .
• Hybrid arrested translation
In this method the portion of mixture is used for in vitro
translation and it serves as the control . The remaining
portion of the mRNA mixture is subdivided and mixed with
denatured recombinant molecule. The mixture is
incubated under suitable conditions favouring annealing .
The DNA insert present in a given clone is hybridize with
the complementary mRNA . The mRNA mixture is used for
in vitro translation and the resulting mixture of
polypeptides is subjected to electrophoresis. The protein
bands obtained in each sample are compared with those
obtained from the control mRNA. The DNA insert causing
the absence of desired protein are identified and isolated.
• Hybrid selection
In hybrid selection method recombinant vectors are
purified ,denatured and fixed separately to a solid
support . The DNA attached to each disc is isolated
separately and used for invitro translation . The
resulting polypeptides are identified by
electrophoresis. The identification of specific
polypeptide may be facilitated by using antibodies
specific to it the antibodies may be used for western
blotting or RNA blotting methods.
• Colony hybridization
This method is used to identify those bacterial colonies in a
petri plate which contain specific DNA sequence .The bacterial
colonies are replica placed or phage plaques are directly lifted
on nitrocellulose filters . The filter disc is removed and put on
blotting paper soaked with 0.5N NaOH solution .The alkali
diffuses into filters, lyses bacterial cells and denatures their DNA
.The disc is neutralized by tris amino methane HCl buffer by
maintaining high concentrations of the salt. The cDNA is fixed
properly by baking at 80 0c. The disc is incubated with a
solution containing radioactive chemical labelled probe at
suitable conditions . The probe hybridizes any bound dna that
contains sequences complementary to probe. The unhybridized
probe is removed by washing. Colonies that develop positive x
ray image are compared with water plate and these colonies are
picked up for further studies.
APPLICATIONS OF rDNA
• Recombinant DNA is widely used in biotechnology,
medicine and research. Today, recombinant proteins
and other products that result from the use of rDNA
technology are found in essentially every western
pharmacy, doctor's or veterinarian's office, medical
testing laboratory, and biological research laboratory.
In addition, organisms that have been manipulated
using recombinant DNA technology, as well as products
derived from those organisms, have found their way
into many farms, supermarkets, home medicine
cabinets, and even pet shops, such as those that sell
GloFish and other genetically modified animals.
• Many additional practical applications of
recombinant DNA are found in industry, food
production, human and veterinary medicine,
agriculture, and bioengineering. Some specific
examples are identified below.
• Recombinant human insulin: Almost completely
replaced insulin obtained from animal sources
(e.g. pigs and cattle) for the treatment of insulin-
dependent diabetes. A variety of different
recombinant insulin preparations are in
widespread use.Recombinant insulin is
synthesized by inserting the human insulin gene
into E. coli, or yeast which then produces insulin
for human use.
• Recombinant human growth hormone:
Administered to patients whose pituitary glands
generate insufficient quantities to support
normal growth and development. Before
recombinant HGH became available, HGH for
therapeutic use was obtained from pituitary
glands of cadavers. This unsafe practice led to
some patients developing Creutzfeldt–Jakob
disease. Recombinant HGH eliminated this
problem, and is now used therapeutically. It has
also been misused as a performance enhancing
drug by athletes and others.
• Recombinant hepatitis B vaccine: Hepatitis B
infection is controlled through the use of a
recombinant hepatitis B vaccine, which contains a
form of the hepatitis B virus surface antigen that
is produced in yeast cells. The development of
the recombinant subunit vaccine was an
important and necessary development because
hepatitis B virus, unlike other common viruses
such as polio virus, cannot be grown in vitro.
Vaccine information from Hepatitis B Foundation
• Golden rice: A recombinant variety of rice
that has been engineered to express the
enzymes responsible for β-carotene
biosynthesis. This variety of rice holds
substantial promise for reducing the incidence
of vitamin A deficiency in the world's
population. Golden rice is not These crops are
in common commercial use in several
• Herbicide-resistant crops: Commercial
varieties of important agricultural crops
(including soy, maize/corn, sorghum, canola,
alfalfa and cotton) have been developed that
incorporate a recombinant gene that results in
resistance to the herbicide glyphosate, and
simplifies weed control by glyphosate
application. These crops are in common
commercial use in several countries
• Insect-resistant crops: Bacillus thuringeiensis is a
bacterium that naturally produces a protein with
insecticidal properties. The bacterium has been
applied to crops as an insect-control strategy for
many years, and this practice has been widely
adopted in agriculture and gardening. Recently,
plants have been developed that express a
recombinant form of the bacterial protein, which
may effectively control some insect predators.
Environmental issues associated with the use of
these transgenic crops have not been fully
Advantages of Recombinant technology:
•Provide substantial quantity
•No need for natural or organic factors
•Tailor made product that you can control
•Resistant to natural inhibitors
Disadvantages of Recombinant technology:
•Effects natural immune system of the body
•Can destroy natural ecosystem that relies on
•Prone to cause mutation that could have harmful
•Major international concern: manufacturing of
biological weapons such as botulism & anthrax to
target humans with specific genotype
• Pharmaceutical biotechnology by Dr .
Chandrakant R. Kokare 9.1-9.22
• Gene biotechnology by S.N Jogdnand 138-
• Internet source