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Название: PCR Protocols: Current Methods and Applications
Автор: White B.
The melding of a technique for repeated rounds of DNA synthesis with the discovery of a thermostable DNA polymerase has given sci-
entists the very powerful technique known as polymerase chain reac-
tion (PCR). PCR is based on three simple steps required for any DNA
synthesis reaction: (1) denaturation of the tern plate into single strands;
(2) annealing of primers to each original strand for new strand synthe-
sis; and (3) extension of the new DNA strands from the primers. These
reactions may be carried out with any DNA polymerase and result in
the synthesis of defined portions of the original DNA sequence. How-
ever, in order to achieve more than one round of synthesis, the templates
must again be denatured, which requires temperatures well above those
that inactivate most enzymes. Therefore, ini tial attempts at cyclic DNA
synthesis were carried out by adding fresh polymerase after each denatur-
ation step (1,2). The cost of such a protocol becomes rapidly prohibitive.