Polymerase Chain Reaction


The polymerase chain reaction (PCR) is a procedure used to amplify DNA, such that large quantities of DNA can be made available from originally small samples. It essentially involves rapid and repeated DNA replication in an artificial environment. PCR is useful in a range of situations: for example, when amplifying DNA samples from crime scenes or from ancient fossils where only trace amounts are available.

A typical PCR reaction mixture contains the DNA sample to be amplified; free deoxynucleotide triphosphate (dNTP) precursors that will be used in replication; a thermostable DNA polymerase enzyme, such as Taq polymerase; and two short oligonucleotides called
primers, each of which should be complementary to either the sense or antisense DNA strand at either end of the region to be amplified. All of this is kept in a buffer solution to stabilize pH and ensure optimum activity of the polymerase enzyme.

The process relies on a series of reactions called thermal cycling. First, the PCR reaction mixture is heated to around 94C for 10-20 seconds to enable the DNA strands to separate by thermally breaking hydrogen bonds (the denaturation step). Then, the temperature is reduced to 50-65C for 20-40 seconds to allow the oligonucleotide primers to anneal to their target sequences on the now-single stranded DNA (the annealing step). The primers should have their 3' ends pointing inwards of the region to be amplified, since the primers will be extended from their free 3' OH group (see notes on DNA replication). Finally, the temperature of the mixture is heated to 72C - the optimum temperature for polymerase activity - and primer extension occurs, whereby free dNTPs are added to the primers to make complementary DNA to the region to be amplified (the extension step).

PCR produces DNA clones exponentially: the first cycle produces 2 DNA molecules from 1, the second cycle produces 4 from 2, the third cycle produces 8 from 4, and so on.