Isolated from the heat-loving bacterium Thermus aquaticus, Taq DNA polymerase launched PCR. It was great, and versions of the original recombinant Taq are still widely used — but its error rate of between 1 in 10,000 and 1 in 50,000 base pairs (bp) is too high for some applications, such as the detection of single-nucleotide mutations. You can now take your pick from a plethora of Taq-based polymerases engineered to have higher fidelity and to go faster. And if damaged DNA is the problem, Restorase from Sigma-Aldrich of St Louis, Missouri, is a mixture of Sigma's AccuTaq blend and a repair enzyme, and works with fragment lengths from 200 to 20,000 bp.

Clean multiplexing with AccuPrime. Credit: INVITROGEN

Developers have also gone back to the planet's hot springs and hydrothermal vents to find a new generation of thermostable polymerases with the 3′–5′ exonuclease proofreading capacity that makes for higher accuracy. The archaeal genus Pyrococcus has been mined for high-fidelity DNA polymerases with accuracies some 40 to 50 times greater than Taq. P. abyssi is the source of the Isis proofreading DNA polymerase from Qbiogene of Irvine, California, with an error rate of one mismatched base per 1.5 million bases per duplication. Other hot offerings come from Stratagene, of La Jolla, California, which claims an error rate of 1 in around 660,000 for its Pfu DNA polymerase from P. furiosus, while Bio-Rad of Hercules, California, has the highly processive iProof High-Fidelity DNA polymerase, a Pyrococcus-type polymerase fused to a double-stranded DNA-binding protein to give additional grip, which the company claims is 50 times more accurate than Taq. As well as a DNA polymerase isolated from P. woesi, Roche Applied Science of Indianapolis, Indiana, offers a thermostable reverse transcriptase from Carboxydothermus hydrogenoformans for RT-PCR, and Toyobo Company of Osaka, Japan, supplies a very fast DNA polymerase from P. kodakaraensis (now renamed Thermococcus).

Proofreading enzymes are generally more finicky than Taq or enzyme blends. To address this problem, Invitrogen's AccuPrime Pfx DNA polymerase is a P. kodakaraensis polymerase in a mix containing the company's proprietary AccuPrime accessory proteins (also available as a mix with the company's Taq polymerase), which improve specificity by ensuring that primers only bind to their complementary sequence. The QuantiTect Multiplex PCR kits from Qiagen, of Hilden, Germany, also aim to provide trouble-free multiplex PCR by including a novel reaction chemistry in the buffer that helps avoid competition between PCR products and ensures efficient primer hybridization.

P.M.