Polymerase chain reaction (PCR)
July 3rd, 2008 | by admin |DNA is mixed with short (10–20 base) single-stranded oligonucleotide primers that are complementary
to the 5′ and 3′ ends of the sequence to be amplified. The mixture is heated to dissociate or “melt” all double-stranded DNA, and then cooled to permit the primers to anneal to their complementary sequences on the DNA to be amplified. Note that the 5′ primer will anneal to the “lower” strand, and the 3′ primer will anneal to the “upper” strand. A heat-resistant (thermostable) DNA polymerase (Taq polymerase, see text) was present in the original mixture, and it now synthesizes DNA by starting at the primers and using the strands to which the primers are annealed as a template. This results in the formation of two double-stranded DNA copies for every molecule of double-stranded DNA in the original mixture. The reaction is then heated to melt double-stranded DNA, cooled to allow reannealing, and the polymerase makes new double-stranded DNA again. There are now four double-stranded DNA copies for each original DNA molecule. This process can be repeated n times (usually 20–50) to result in 2” copies of double-stranded DNA.
