The following enzymes are used in vitro when performing recombinant DNA techniqu

Question

The following enzymes are used in vitro when performing recombinant DNA techniques.
They can be readily purchased from biotechnology companies who prepare them. Describe
where biotechnology companies get the following enzymes from and then describe the use of
each enzyme in recombinant DNA techniques. Don’t simply state the function of the enzyme but
indicate WHY or HOW we use the enzyme.
(a) Restriction Enzymes (RE):
Source:
Use:
(b) DNA Ligase:
Source:
Use:
(c) Taq DNA polymerase:
Source:
Use:
(d) Reverse Transcriptase (RT):
Source:
Use:

Explanation / Answer

Nowadays, recombinant DNA technology is used to produce most of the enzymes. Earlier it was produced by growing the particular bacteria and then extracting the enzyme. rDNA technology now offers the cloning of various enzyme coding gene in E. Coli and then enzymes produced are purified. This is how the biotechnological companies get the enzymes. The method is used for producing variety of enzymes- restriction enzymes, DNA ligase, Taq DNA polymerase, reverse transcriptase.

The above statements are true for all the following enzymes, difference lies in the gene used. I am listing the gene to corresponding enzyme.

(a) Restriction Enzymes (RE):

Source: Genes coding for various restriction enzymes isolated from corresponding bacteria.
Use: Restriction enzymes are used to create breaks in dsDNA at specific sites. These sites are palindromic hence produce sticky ends which allows the insertion of foreign DNA fragment at the site where the 'break' is created. Restriction enzymes are site specific. Therefore restriction enzymes are the most important tool in rDNA technology.
(b) DNA Ligase:
Source: Genes coding for DNA ligase enzyme isolated from bacteria.
Use: DNA ligase is the enzymes used for filling the gaps between DNA strands.
(c) Taq DNA polymerase:
Source: Genes coding for DNA polymerase enzymes isolated from bacteria Thermus aquaticus - a bacteria native of hot spring. Hence the T. aquaticus produce thermostable DNA polymerase enzyme.
Use: Taq DNA polymerase is used in in vitro DNA polymerization of DNA. It polymerizes the target DNA in thermal cycler when provided with suitable reagents and reaction conditions (PCR). It is the core component of the PCR (Polymerase Chain Reaction).
(d) Reverse Transcriptase (RT):
Source: Genes coding for reverse transcriptase.
Use: Reverse transcriptase (RT) synthesizes DNA using RNA as template. The enzyme is extremely useful in gene expression studies where RNA is template. Various molecular biology experiments needs RT enzyme.

All above enzymes are the prerequisite for any molecular biology experiment. Its production on commercial level using rDNA technology has really proved a great advantage to molecular biologists.

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