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Question
What is PCR? Explain different steps involved in it.
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Solution
Definition: Polymerase Chain Reaction (PCR) is the process of in vitro amplification of gene of interest using a PCR machine.
Mechanism of PCR: At the start of PCR, all the requirements are mixed together in ‘eppendorf tube’ and the following operations are performed sequentially:
Step i: Denaturation
The reaction mixture is heated to a temperature (90–98o C) to separate two strands of desired DNA. This is called denaturation.
Step ii: Annealing
The mixture is allowed to cool (40–60o C) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing.
Step iii: Primer extension / Polymerization
The temperature (70–75o C) allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides. This is called primer extension. It takes around two minutes duration.
RELATED QUESTIONS
Rearrange the following in the correct sequence to accomplish an important biological reaction:
(a) In vitro synthesis of copies of DNA of interest
(b) Chemically synthesized oligonucleotides
(c) Enzyme DNA-polymerase
(d) Complementary region of DNA
(e) Genomic DNA template
(f) Nucleotides provided
(g) Primers
(h) Thermostable DNA-polymerase (from Thermus aquaticus)
(i) Denaturation of ds-DNA
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