Comparing the Speed and Product Yield of 7 high fidelity DNA Polymerases

Thermococcus kodakaraensis (strain KOD1), previously thought to be a Pyrococcus sp., is a hyperthermophilic archaea isolated from a solfataric hot spring on Kodakara Island, Japan (Atomi 2004). In preliminary studies to characterize the KOD1 DNA polymerase, researchers found that the enzyme had fidelity comparable to Pfu DNA polymerase, but with an extension rate (referred to as speed) 5 times higher and processivity 10 to 15 times higher than the Pfu enzyme (Takagi 1997). When amplifying DNA for cloning, high fidelity DNA polymerases, such as KOD polymerase, are recommended. If the enzyme is also fast and can generate high yields of full-length product, fewer amplification cycles are required and the probability of obtaining error-free clones is greatly increased. Since the preliminary KOD DNA Polymerase studies, significant work has been done to optimize the PCR buffer and cycling parameters. Another improvement is KOD Hot Start DNA Polymerase, which is a premixed complex of KOD DNA Polymerase and two monoclonal antibodies. The antibodies inhibit the 3 ́→5 ́ exonuclease and DNA polymerase activities at ambient temperatures (Mizuguchi 1999), providing high template specificity by preventing primer degradation and mispriming events during reaction set-up. This report evaluates the speed and product yield of KOD Hot Start DNA Polymerase in an optimized reaction buffer and compares the enzyme to 6 other commercially available high fidelity thermophilic DNA polymerases.