The polymerase chain reaction
The development of the polymerase chain reaction (PCR)
technique has revolutionised molecular genetics. This
technique uses two short single strand DNA primers
(typically 20 bp to 25 bp long) to initiate DNA replication
at a specific point on the DNA molecule. A thermostable
DNA polymerase is then used to copy the DNA by
extending the primers and synthesising complementary
strands of DNA. Repeatedly denaturing the DNA,
reannealing the primers, and copying the DNA will
exponentially amplify the target sequence between the
primers. Following the amplification process, sufficient
DNA is available for analysis directly by electrophoresis.
The PCR-amplified DNA can be digested with a restriction
enzyme and visualised by gel electrophoresis to determine
if the PCR fragments have been cleaved. Polymerase chain
reaction and PCR-RFLP are frequently used in diagnostic
testing to determine the genotype at a known genetic
mutation (Fig. 2).