High-Fidelity DNA Polymerase comparison

High-Fidelity DNA Polymerase Comparison on difficult genomic DNA samples.

Modern commercially available high-fidelity DNA Polymerases are derived from either Pfu and KOD. Pfu is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus. KOD is an enzyme isolated from Thermococcus kodakaraensis. Both original enzymes have now been genetically engineered to offer better processing, lower mutation rates and faster extension rates. A modern trend in the market is to fuse the polymerases with a DNA binding domain such as the Sso7d DNA-binding domain in order to increase affinity for DNA and prevent the polymerase from ‘dropping-off’ the DNA template during DNA extension, thereby enhancing processivity and sometimes even fidelity. Such a fusion protein became very popular when Finnzymes commercialized the polymerase Phusion® (now owned by Thermo Fisher Scientific). Phusion® and Q5® DNA polymerases (the latter is a trademark of New England Biolabs) are considered as the most advanced polymerases on the market in 2016. A new generation of high-fidelity DNA polymerases is now emerging slowly. These are not fusion proteins, are very processive and do not require extreme PCR cycling conditions or even high primer concentrations such as in the case of Phusion and Q5 high-fidelity DNA polymerases. Hence, we perfromed a High-Fidelity DNA Polymerase Comparison using these novel DNA Polymerases.

For some templates or depending on the DNA extraction method, finding a high-fidelity enzyme (or even a regular Taq DNA polymerase) that can be versatile and perform under harsh conditions can be a challenge. For instance, Phusion® and Q5®’s recommended annealing temperatures for primers and DNA template can prove favorable in some instances but can also be problematic if the required annealing temperature exceeds that of the extension step during PCR cycling. High annealing temperatures, along with a 2-step PCR protocol (annealing and extension steps combined), augments the probabilitily of non-specific amplification. In such instances, there is no room for PCR optimization at all, but redesign the primers themselves. Phusion® and Q5® are said to obey to the Tm + 3 °C rule to determine the optimal annealing temperature (Ta) used in PCR cycling. In contrast, the general rule for Taq DNA Polymerase is Tm -5 °C.

The TransDirect® Animal Tissue PCR Kit uses a proprietary buffer in which samples are incubated for 10 min at room temperature and then heated 3 min at 95 °C. Lysates are then normally combined with 2x TransDirect PCR supermix containing a special inhibitor-resistant DNA polymerase (TransBD) and specific primers to perform PCR. The whole process is advantageous for high-throughput labs becuse it requires much less time and ressources (no centrifugation) and less pipetting steps than column-based gDNA extraction.The TransDirect Animal PCR kit was successfully used in another post (Direct PCR on hair, saliva, cheek scrub and dead skin lysates) to lyse cells from hair, saliva, cheek and dead skin.

Our goal was to perform a High-Fidelity DNA Polymerase Comparison under more difficult conditions. According the respectuve datasheets, TransStart FastPfu and TransStart FastPfu FLY, obey to the Tm -5 °C, rule with leading polymerases from competitors, namely Phusion® Hotstart II (M549; ThermoFisher Scientific) and Q5® high-fidelity DNA Polymerase (M0491; New England Biolabs) use Tm + 3 °C. Tests were carried on TransDirect® Animal Tissue kit lysates (performance described here) and on raw extracts of (unpurified) HeLa and 293 cell lysates.