18-08-2012, 02:39 PM
Polymerase chain reaction(PCR)
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Defination
Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.
This method was first time developed by Kary Mullis in 1983.
It is a very simple and inexpensive technique for characterizing , analyzing and synthesizing any specific piece of DNA or RNA from virtually any living organism.
Principle:
PCR works on the basic principle of three temperature cycles:
Denaturation(92°c-96°c)-two strands melt open to form ssDNA.
Annealing(45°c -55°c)-annealing of primers to each original strand for new strand synthesis.
Extension.(72°c)-polymerase adds dNTPs complementary to the template at the 3’ end of the primers , since both strands are copied during PCR there is an exponential in in the no. of copies of the genes.
DNA
DNA is a nucleic acid that is composed of two complementary nucleotide building block chains.
The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base.
Generally nanogram or picogram amount of DNA is used.
Higher amount of DNA inhibits or results in non specific amplification.
PCR Primers
Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the
complementary building blocks of the target
sequence.
A primer for each target sequence on the end
of your DNA is needed. This allows both
strands to be copied simultaneously in both
directions.