Steps involved in Recombinant DNA technology
- Selection and isolation of DNA insert
- Selection of suitable cloning vector
- Introduction of DNA-insert into the vector to form a rec DNA molecule
- rec DNA molecule is introduced into a suitable host.
- Selection of transformed host cells.
- Expression and multiplication of DNA-insert in the host.
- Selection and isolation of DNA insert:
The first step in rec DNA technology is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.
- Selection of suitable cloning vector:
A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rec DNA technology. The most commonly used vectors are plasmids and bacteriophages.
- Introduction of DNA-insert into the vector to form recDNA molecule:
The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes [in step (i)] are now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA molecule which is often called as cloning vector-insert DNA construct.
- rec DNA molecule is introduced into a suitable host:
Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)] is introduced into these host cells. This process of entry of rec DNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however, yeast, fungi may also be utilized.
- Selection of transformed host cells:
Transformed cells (or recombinant cells) are those host cells which have taken up the recDNA molecule. In this step, the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.
- Expression and Multiplication of DNA insert in the host:
Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain a sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.
Tools for Recombinant DNA Technology:
Important biological tools for rec DNA technology are:
- Restriction Endonucleases
- DNA ligases
- DNA polymerase (B) Cloning Vector
- Host organism
- DNA insert or foreign DNA
- Linker and adaptor sequences.