The polymerase chain reaction (PCR) is a test tube system for DNA replication which allows a “target” DNA sequence to be selectively amplified several million fold in just a few hours. The PCR achieves amplification of a predetermined fragment of DNA, (the target; which can e.g. be from 100 – 1000 bp long)
A basic PCR set up requires several components and reagents. These components include:
- DNA template that contains the DNA region (target) to amplify
- Taq polymerase, a DNA polymerase that is heat resistant, so that it can remain intact during the DNA denaturation process.
- Two primers that are complementary to the 3' ends of each of the sense and anti-sense strand of the DNA target (DNA polymerase can only bind and elongate from a double-stranded region of DNA, and without primers there is no double-stranded initiation site for polymerase to bind).
- Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
- Bivalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis
- Monovalent cation potassium ions
|Typically, PCR consists
of a series of 20–40 repeated temperature
changes, called cycles, with each cycle commonly consisting of 2–3
discrete temperature steps. The cycling is often preceded by a single
temperature step at a high temperature (>90 °C), and followed by
hold at the end for final product extension or brief storage. The
temperatures used and the length of time they are applied in each cycle
depend on a variety of parameters. These include the enzyme used for
DNA synthesis, the concentration of divalent ions and dNTPs in the
reaction, and the melting temperature (Tm) of the primers.