Variants of PCR - Other Modifications

Other Modifications

Adjustments of the components in PCR is commonly used for optimal performance:

• The divalent magnesium ion (Mg++) is required for PCR polymerase activity. Lower concentrations Mg++ will increase replication fidelity, while higher concentrations will introduce more mutations.

• Denaturants(such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Other chemicals, such as glycerol, are stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent polymerase stick to itself or to the walls of the reaction tube.

• DNA polymerases occasionally incorporate mismatch bases into the extending strand. High-fidelity PCR employs enzymes with 3'-5' exonuclease activity that decreases this rate of mis-incorporation. Examples of enzymes with proofreading activity include Pfu; adjustments of the Mg++ and dNTP concentrations may help maximize the number of products that exactly match the original target DNA.

• COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a modified Polymerase Chain Reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA.