5 Steps in Gene cloning

What is Gene Cloning?

Gene Cloning or molecular cloning is the process of making many identical copies of a DNA fragment or gene.

Applications of Gene cloning

  • Study of gene structure and function
  • If it is a newly studied gene; Gene cloning followed by DNA sequencing helps us to understand the structure of the gene.
  • Mutagenesis to understand the function of the cloned gene.
  • Expression of cloned gene is preferred if the gene codes for some therapeutically important proteins like insulin.

5 Steps in Gene cloning
5 Steps in Gene cloning

Step 1: Identification & Isolation of Gene of interest or DNA fragment to be cloned.

Step 2: Insertion of this isolated gene in a suitable vector.

Step 3: Introduction of this vector into a suitable organism/ cell called host (transformation).

Step 4: Selection of the transformed host cell.

Step 5: Multiplication or expression of the introduced gene in the host.

What are the steps in Gene cloning

Step1: Identification and isolation of gene of interest

From where we get the desired gene?

If we are studying a gene or a DNA fragment for the first time, we need to construct a genomic library


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 by forming phosphodiester bond. 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. The process is called transformation

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 imbibition by cells, tissues or organs: Transformation

d. Agrobacterium mediated gene transfer in plants

e. 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:

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 desired gene. The inserted gene along with the vector will replicate inside the host so that many copies of the desired gene is synthesized. This process of making many copies of the desired gene or DNA fragment is called gene cloning.

For expression of the desired gene, expression vector is used (vector with control elements like promoter, operator etc. The product is synthesized in bioreactors; large culture vessels for large scale production.

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