polymerase chain reaction. The amplification of a specific DNA sequence, termed target or template sequence, that is present in a complex mixture, by adding two or more short oligonucleotides, also called primers, that are specific for the terminal or outer limits of the template sequence. The template-primers mixture is subjected to repeated cycles of heating to separate (melt) the double-stranded DNA and cooling in the presence of nucleotides and DNA polymerase such that the template sequence is copied at each cycle. Thermostable polymerases such as those obtained from a hot springs bacterium Thermus aquaticus, commonly termed Taq polymerase, are used.
At the end of 20 to 30 such cycles, the amplified target sequence, which may have been present in as few as a single copy in the original mixture, can be readily detected, for example by electrophoresis and ethidium bromide staining in an argarose gel.
the detection of more than one template in a mixture by addition of more than one set of oligonucleotide primers.
the primers used in the first round of amplification are either both replaced (nested PCR) or only one is replaced (semi-nested PCR) for the second and subsequent cycles of amplification. Increases the sensitivity and specificity of the PCR.
a means for quantifying the amount of template DNA present in the original mixture. Usually achieved by the addition of a known amount of a target sequence that is amplified by the same primer set but can be differentiated, usually by size, at the end of the reaction.
a method for the detection and quantitation of an amplified PCR product based on incorporation of a fluorescent reporter dye; the fluorescent signal increases in direct proportion to the amount of PCR product produced and is monitored at each cycle, 'in real time', such that the time point at which the first significant increase in the amount of PCR product correlates with the initial amount of target template.
reverse-transcriptase PCR (RT-PCR)
a reaction applied when the target sequence is RNA, such as viral RNA or messenger RNA. Reverse transcriptase that copies DNA from an RNA template is present in the first round.