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Steps in Recombinant DNA
technology or Gene cloning
Let us start with definition
Definition: 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
and production of large quantities of
that gene fragment or product
encoded by that gene.
An Example of a product synthesized
using rDNA technology
Humulin, is insulin developed using
rDNA technology which is used to
treat diabetes. Here insulin is
synthesized inside bacterium where
we introduced human insulin gene.
Thus bacterial system just works as
biofactories for the synthesis of
insulin.
How this is achieved? We will be
discussing the basic steps involved in
rDNA technology, gene cloning or
genetic engineering.
Step1: Identification and isolation of
gene of interest
From where we get the desired
gene?
From
 Genomic library
 cDNA library
 Chemical synthesis of gene if
we know the sequence
 If the number of copies of the
desired gene is not enough for
gene cloning we can opt for
gene amplification techniques
like PCR
Step II: joining of this gene into a
suitable vector (construction of
recombinant DNA)
What is a Gene Cloning Vector?
A vector is any DNA molecule which
is capable of multiplying inside the
host to which our gene of interest is
integrated for cloning. The selection
of vector depends upon the size of
the fragments to be cloned.
Common vectors include plasmids
(Eg: pBR 322) and phage vectors.
In the process, restriction
enzymes functions as scissors for
cutting DNA molecules. Ligase
enzyme is the joining enzyme that
joins the vector DNA with gene of
inertest. The resulting DNA is called
the recombinant DNA, chimera or
recombinant vector.
Step III: Introduction of this vector
into a suitable organism
Introduction of recombinant vector
into host cell is achieved by
different gene transfer methods
a. Physical gene transfer methods:
 Electroporation
 Microinjection
 Liposome mediated gene
transfer
 Silicon Carbide fibre mediated
gene transfer
 Ultrasound mediated gene
transfer
 DNA transfer via pollen
b. Chemical gene transfer methods:
 Poly Ethylene Glycol mediated
(PEG mediated),
 Calcium Chloride mediated
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 DEAE dextran mediated gene produced in large quantities in cell
transfer cultures.
c. DNA imbibitions by cells, tissues or
organs: Transformation
d. Virus mediated gene transfer:
Transduction

Step VI: Selection of transformed

recombinant cells with gene of
interest
The number of cells with
recombinant vector will be very less.
So the next step is to select the
transformed recombinant cells with
Youtube Video on
our gene of interest from the sea of
Steps in Recombinant DNA
non transformed cells. Several
technology or Gene cloning
methods are employed for selection
of transformed cells:

 Antibiotic resistance,
 Visible characters,
 Assay for biological activity,
 Colony hybridization,
 Blotting test.
 The selected cells are cultured
in large scale.
Step V: Multiplication or expression
of the gene of interest
The objective of gene cloning is either
to make numerous copies of the
desired gene or to produce the
protein coded by the desires gene.
The inserted gene along with the
vector will replicate inside the host so
that many copies of the desired gene
is synthesized.
For expression of the desired gene,
expression vector is used (vector with
control elements like promoter,
operator etc). The product is
synthesized in mass cultures in large
quantities. This is how insulin is