Question

Explain the various steps involved in recombinant DNA technology.

Answer 1

Recombinant DNA technology involves several important steps that include:

1. Isolation of Gene of Interest: The first step in rDNA technology is isolating the needed DNA in its pure state, devoid of extraneous macromolecules. To separate and purify DNA from other macromolecules in the cell membrane, enzymes including lysozymes, cellulase, chitinase, ribonuclease, and proteases are used, among others.
2. Restriction Enzyme Digestion: After purifying the DNA, restriction enzymes isolate the desired gene. Restriction enzymes act as molecular scissors, cutting DNA at specified sites. In this stage, pure DNA is incubated with the specified restriction enzyme under ideal conditions. The procedure for processing vector DNA remains the same.
3. Amplification Using PCR: Polymerase Chain PCR is a technique for creating numerous copies of a DNA sequence using the enzyme DNA polymerase in vitro. Amplifying a single or a few copies of DNA can result in thousands or millions of copies. Cut DNA fragments can be amplified using PCR and ligated to the cut vector.
4. Ligation of DNA Molecules: The pure DNA and vector of interest are cut with the same restriction enzyme and then ligated with ‘DNA ligase’. The generated DNA molecule combines the interest molecule and the vector. The novel hybrid DNA molecule is also known as recombinant DNA.
5. Insertion of Recombinant DNA into Host: In this stage, recombinant DNA is introduced into recipient host cells, often bacterial cells. This process is known as transformation. Hosts are treated to enable them to accept new DNA. The procedures used may include thermal shock, Ca²⁺ ion treatment, electroporation, and others.
6. Isolation of Recombinant Cells: The transformation process generates a mixture of transformed and non-transformed cells. The selection procedure solely filters transformed host cells. Gel electrophoresis is a technique for separating DNA and identifying plasmids that contain the desired gene. Several more ways include culturing cells on antibiotic (e.g., tetracycline) agar plates.