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PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction)


PCR
The polymerase chain reaction, or PCR, has become one of the most widely used techniques in modern molecular biology. The uses of DNA amplification are so many and varied that in addition to simple PCR a host of variations of the technique are now used in almost every discipline from forensic pathology to infectious disease epidemiology.

The development of PCR hinges around the isolation of a DNA polymerase from a thermally stable microorganism called Thermus Aquaticus, or Taq which was found in a hot spring in Yellowstone National Park. This can be used successfully at temperatures above 75oC.

The structure of the DNA molecule also lends itself very well to PCR. In their original Nobel Prize-winning paper, Watson and Crick commented: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”. Indeed, as DNA is heated above a certain temperature, the two strands ‘unzip’. And, as the temperature cools back down, the nucleotides attached to the phosphate backbone find each other again and the molecule is able to reform back into its original structure. It is this property which is used throughout the PCR technique. 

In simple terms, the starting points are a solution which consists of the purified DNA containing the segment which is to be amplified, a thermally stable polymerase, such as the Taq polymerase already mentioned and a mixture of free nucleotides and optimised buffers. In addition to this, primers are added to kick start the reaction. These are oligonucleotides which match a known sequence of the gene or region of DNA which needs to be targeted. 

A single thermal cycle consists of roughly three steps in which the temperature of the whole solution is first raised, held, then lowered. These steps consist of denaturation of the DNA strand, annealing, or attaching of the primers and nucleotides, and then extension. This is the crucial point when the polymerase is able to extend the DNA molecule.

A full PCR cycle might contain between 30 and 40 complete cycles. At each end point, there should be roughly double the amount of DNA than there was at the start. This rapid growth means that there are literally millions more molecules in solution at the end of the PCR reaction. Once complete, the sample can be run on an electrophoresis gel and compared with a DNA mixture of known sizes, called a ladder because of the way the molecules distribute in the gel. Using this technique very tiny amounts of DNA can be amplified and examined.

More information on Reverse Transcription PCR can be found at this link.