Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is another key device used for gene cloning and gene multiplication in vitro -- it amplifies a chosen gene of interest. In 1985, Kary Mullis made the important discovery of this extremely powerful technique. PCR can generate a billion copies of a desired DNA or RNA segment, with high accuracy and specificity, in a matter of hours. The whole process is completely automated and runs through repeated thermal cycles of denaturation and renaturation of double-stranded DNA inside a machine called a thermal cycler.

PCR requires: the DNA containing the segment to be amplified, an excess of the four deoxyribonucleoside triphosphates (dNTPs), an excess of two primer molecules, a heat-stable DNA polymerase, and an appropriate quantity of Mg2+ ions. One cycle takes roughly 3 to 4 minutes, and to begin the next cycle the DNA is again heated to convert it back to single strands. In an automatic thermal cycler, the three steps below repeat automatically 20 to 30 times, so that after 'n' cycles, 2^n copies of the DNA segment have been produced -- entirely automatically and with high precision. Once the desired number of cycles is complete, the amplified segment is purified by gel electrophoresis; after sequencing, it can be inserted into a cloning vector (a desired gene can also be sourced directly from a gene library).

The mechanism of PCR (illustrated in Fig 12.1) runs through three steps every cycle, with the DNA segment, excess primers, the four dNTPs and the thermostable DNA polymerase all mixed together in a small reaction (eppendorf) tube:

Step i -- Denaturation. The reaction mixture is heated to 90-98 degrees C, which separates the two strands of the target DNA.

Step ii -- Annealing. The mixture is cooled to 40-60 degrees C, which lets the two primers pair with their complementary sequences on the separated DNA strands. …