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Steps in Recombinant DNA Technology or rDNA Technology

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.
Steps in Recombinant technology
Step1: Identification and isolation of gene of interest
From where we get the desired gene?
  • 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 interest. 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
  • DEAE dextran mediated gene transfer
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 our gene of interest from the sea of non transformed cells. Several methods are employed for selection of transformed cells:
Result of transformation
  • 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 produced in large quantities in cell cultures.
Steps in Recombinant DNA technology or rDNA technology PDF Click here
We will be discussing each of the techniques mentioned in the above steps in detail in the coming posts
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