PCR (2): Methodology
PCR has been performed on DNA larger than 10 kilobases, however the average PCR is only several hundred to a few thousand bases of DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Usually, the longer the fragment, the greater the probability of errors.
PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments, usually up to 10 kb. Certain methods can copy fragments up to 47 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs.
PCR, as currently practiced, requires several basic components. These components are:
- DNA template, which contains the region of the DNA fragment to be amplified.
- Two primers, which determine the beginning and end of the region to be amplified.
- Taq polymerase, a DNA polymerase, which copies the region to be amplified
- Deoxynucleotides-triphosphate, from which the DNA Polymerase builds the new DNA
- Buffer, which provides a suitable chemical environment for the DNA Polymerase
The PCR process is carried out in a thermal cycler. The reaction tubes are heated and cooled to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top on the cycler.
