PCR and DNA Amplification

Taq DNA polymerase belongs to polymerase Family A, a group of related enzymes that comprises bacterial repair polymerases and most bacteriophage replicative polymerases. Family A polymerases generally have both replicative and repair activities. The important “proofreading” function that allows repair of a mis-paired nucleotide is provided by 3’→5’ exonuclease activity, while 5’→3’ exonuclease activity allows the removal of RNA primers. In Taq polymerase, the 3’→5’ exonuclease proofreading domain has been changed and is not functional.

PCR imposes the same basic demands on a polymerase as a cell puts on its replication system: reliability, accuracy, or fidelity, speed and processivity. Polymerase accuracy, or fidelity, refers to the incorporation of the correct nucleotides as specified by the template strand. DNA polymerases ensure accurate replication using a series of molecular checkpoints at the site of nucleotide incorporation. Standard Taq polymerase is quite accurate with a relatively low total error rate but lacks 3’→5’ exonuclease “proofreading” activity and this limits the enzyme’s effective possible amplicon size. Taq performs best, and is a robust, easily optimized enzyme, when amplifying DNA fragments <2 kb. The enzyme can work with fragments up to 3–4 kb but efficiency quickly drops with amplicons longer than about 3 kb.

Hot-start PCR is facilitated through modifications to the DNA polymerase that block amplification by rendering the enzyme inactive until a higher temperature is reached. Common modifiers include antibody interaction, chemical modification, and aptamer technology. When permissive reaction temperatures are reached during PCR cycling, the inhibitor dissociates from the polymerase and polymerization commences.

Enzyme-linked anti-Taq DNA polymerase antibodies inactivate the enzyme by linking and binding to the polymerase, preventing early DNA amplification at lower temperatures. Once the optimal annealing temperature is met, the antibodies begin to degrade and dissociate, releasing the Taq DNA polymerase into the reaction and allowing the amplification process to start.

Chemically modified Taq DNA polymerases have heat-labile blocking groups attached to specific amino acid residues. These are removed by an initial high temperature activation step in the PCR reaction. This restores the Taq DNA polymerase to full activity and PCR cycling can proceed.
Aptamer-mediated hot-start PCR relies on inhibiting the function of the Taq DNA polymerase through the attachment of oligonucleotide aptamers. Aptamers dissociate from the enzyme at a lower temperature than heat-labile chemical blocking groups or antibodies, thereby eliminating the need for a high temperature activation step and accelerating PCR protocols. In addition, aptamer-based inhibition is fully reversible so that Taq DNA polymerase activity is also blocked at the end of thermal cycling.