question_answer

With the help of diagrams show different steps involved in the cloning and expression of a y gene responsible for the production of growth hormone into E. coli.

Or

If a desired gene is to be identified in an organism for some specific purpose, explain the process of the following.

(i) Cutting the desired gene at specific location.

(ii) Synthesis of multiple copies of this desired gene mentioning all required steps.

Answer:

The multiple identical copies of a specific template DNA can be produced by DNA cloning. It can be carried out by the use of a vector to carry specific foreign DNA fragment into the host cell. The mechanism of cloning and transfer of a gene for human growth hormone into E. coli is diagrammatically represented below

Diagram showing steps involved in cloning and expression of a growth hormone gene into E coli

Or

In order to identify a desired gene in an organism for some specific purpose, the process given below will occur in the following way

(i) Cutting of desired gene at specific location Digestion of DNA molecules can be performed by incubating purified DNA molecules with the restriction enzyme. This is done at optimal conditions specific for that enzyme. Agarose gel electrophoresis is used to check the progress of restriction enzyme digestion.

(ii) Synthesis of multiple copies of the desired gene This can be done by using PCR (Polymerase Chain Reaction). It is also known as in vitro replication of DNA. By using PCR, amplification of gene of interest can be easily done under three main steps, i.e.

(a) Denaturation The dsDNA is denatured by using high temperature of \[95{}^\circ C\] for 15 sec. Each separated ssDNA now acts as a template for the further synthesis of DNA.

(b) Annealing This can be done by using two sets of oligonucleotide primers. This step is performed at somewhat lower temperature\[(40-60{}^\circ C)\]using \[M{{g}^{2+}}\] as a cofactor for Taq polymerase and dNTPs depending upon the length and sequence of the primers.

(c) Extension The thermostable enzyme, i.e. Taq polymerase is used in the reaction as it can tolerate the high temperature and can extend the   primers   by   adding   nucleotides complimentary to the template.