DNA Science

Polymerase Chain Reaction

PCR is an acronym, which stands for polymerase chain reaction. The PCR technique is a primer extension reaction for amplifying specific regions of DNA. PCR allows a stretch of DNA to be amplified to about a million fold so that its nucleotide sequence can be determined.

The particular stretch of DNA to be amplified, called the target sequence, is identified by a specific pair of DNA primers. These primers, usually about 20 nucleotides in length, hybridise to opposite strands of the double stranded DNA and flank the region of interest. With the addition of a DNA polymerase the target DNA is replicated and the amount of DNA is increased.

A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase, results in the exponential growth of the target DNA sequence.

Real time PCR (or quantitative PCR (Q-PCR)) is the ability to monitor the progress of the PCR as it occurs i.e. in real time. The data is therefore collected after every PCR cycle throughout the PCR process, rather than at the end of the PCR.

Real time PCR uses the point in time that the amount of DNA is first detected and enters the exponential phase, rather than the end point amount, to determine the concentration of a sample. The larger the starting amount of target DNA, the earlier (in number of cycles) the exponential phase is reached. This is observed as a large increase in fluorescence. Using a standard curve the amount of DNA in a particular sample can be calculated.

The real time PCR method is based on the detection and quantification of a fluorescent molecule. The amount of fluorescence increases in direct proportion to the amount of the target DNA in the reaction. By recording this fluorescent emission at the end of each cycle, it is possible to monitor the PCR reaction and therefore the amount of DNA present.