Gene manipulation and recombinant DNA technology - Pharmaceutical Biotechnology: Concepts and Aapplications - page 54

5' 3'

3' 5'

Add primers & other reaction constituents,
heat & then cool to allow primers to anneal

+

Extension phase

+

Repeat heating & cooling cycles

Figure 3.15 The PCR is initiated by separation of the double-stranded DNA into its two constituent strands.
This is achieved by heating the sample (usually to 94 C). Also present in the reaction mixture are: (a)
two chemically synthesized oligonucleotide primers ('oligos') whose sequences are complementary to the
sequences fl anking the gene of interest; (b) the enzyme DNA polymerase, which can extend the primers to
synthesize a new DNA strand of complementary sequence to the single stranded DNA template; (c) all the
nucleoside precursors required for synthesis of the growing DNA strand (i.e. the deoxynucleoside triphos-
phates or dNTPs). Once strand separation has been achieved the reaction temperature is reduced, in order to
allow primers to anneal to complementary sequences on each strand and allow the DNA polymerase to extend
the primers. This extension phase is normally carried out at 74 C. The DNA polymerase used is sourced from
the thermophilic microorganism Thermus aquaticus ; therefore, it is heat stable and not inactivated by PCR
operational temperatures. This completes the fi rst cycle of the PCR process; the result is a doubling of the
amount of target DNA present in the reaction mixture. The cycle is then repeated and with each repeat comes
a doubling of the amount of target DNA. After 25-30 cycle repeats, several hundred million copies of the
target DNA have been generated