Conventional PCR

Conventional PCR

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 one 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.

  • Initialization step (Only required for DNA polymerases that require heat activation by hot-start PCR): This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes.
  • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
  • Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template.
  • Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to amplify.