Editorial Article
Friday December 11, 2009
by Caitlin Smith
Taq DNA Polymerase, an enzyme that catalyzes the incorporation of dNTPs into DNA, is one of the most commonly used polymerases in molecular biology. Though it lacks proofreading ability, and other polymerases have since been developed, ¡§Taq DNA polymerase has been a workhorse for PCR for many years,¡¨ says Andrew Gardner, a research associate at New England Biolabs. As such, this article focuses on high-tech developments of Taq itself. Because according to Daniel Corey, director of sales and marketing at Bioline, ¡§it is clear that researchers are asking more complex questions than ever and continue to rely on the PCR technique and Taq polymerase.¡¨
Different flavors of Taq
Some Taq vendors try to set their product apart. ¡§New England Biolabs [provides] one of the largest ranges of polymerase options targeted at the ever-growing list of PCR applications and different starting samples used,¡¨ says Gardner. ¡§In the future, there will also be increased demand to amplify long DNA fragments. Taq DNA polymerase can routinely amplify fragments up to 10 kb. NEB¡¦s LongAmpTM Taq DNA polymerase, however, has been optimized to amplify a broad range of DNA amplicons from less than 1kb up to 40 kb.¡¨
Bioline also offers a Taq suitable for long fragments. ¡§PCR has become more complex in that researchers are amplifying long fragments or problematic sequences which contain high GC content,¡¨ says Corey. ¡§Bioline has addressed this trend by developing and offering Taq polymerase blends, which include proofreaders for increased processivity. Bio-X-Act Long and Short DNA Polymerases are well-established examples of PCR blends designed for problematic or difficult PCR.¡¨
Applied Biosystems, part of Life Technologies, offers another example of high-tech Taq products in their new AmpliTaq Gold 360 DNA Polymerase and Master Mix, for use in hot-start PCR reactions. ¡§This product is delivered with the 360 GC Enhancer, which allows you to work through challenging GC-rich sequences,¡¨ says Carol Dascomb, marketing manager for PCR systems at Life Technologies. ¡§Compared with alternative enzymes, this product works more often for more template types, saving the researcher valuable time, sample, and enzyme.¡¨
Reducing steps for using Taq
Eliminating unnecessary steps in the PCR workflow saves time and reagents, while minimizing contamination. Gardner thinks that the ability to perform extraction-free PCR directly from tissue samples is one of the most exciting developments in Taq polymerase products today. ¡§Amplification of DNA directly from starting materials such as blood or tissue has been problematic due to the presence of inhibitors in the sample,¡¨ says Gardner. ¡§As an alternative [to purification of genomic DNA], NEB has recently made available several kits and polymerases that can be used for the direct amplification of DNA, without the need for purification steps.¡¨
For example, NEB¡¦s Hemo KlenTaq„· is a truncated version of Taq DNA polymerase with mutations that make it resistant to inhibitors in whole blood. ¡§Hemo KlenTaq offers the versatility and robustness of Taq, but can successfully amplify samples containing up to 10% whole blood,¡¨ says Gardner. In addition, NEB¡¦s Crimson TaqTM DNA polymerase, in a reaction buffer containing trace amounts of a red indicator dye, makes gel loading and tracking easier.
Bioline¡¦s MangoMix also contains colored dyes for gel-loading, in addition to Taq polymerase, ultra-pure dNTPs, reaction buffer, MgCl2, and enhancers. ¡§There is a clear demand for more convenient formats such as 2X PCR mastermix, [MangoMix],¡¨ says Corey. ¡§Bioline has long been established as a primary manufacturer of UltraPure dNTPs and in our experience, the quality of the dNTPs is very important for critical assays such as long PCR and qPCR.¡¨
Taq limitations and challenges
Gardner says that a limitation in using Taq polymerase is its low fidelity rate compared to other types of polymerases. It is possible, however, that a form of Taq with a higher fidelity rate could be developed. ¡§We anticipate a growing demand for Taq DNA polymerase variants that incorporate modified nucleotides more effectively for DNA detection and labeling,¡¨ he says. ¡§NEB has expanded its DNA polymerase screening program to select for Taq DNA polymerase variants with desired properties.¡¨
Dascomb says that the routine task of optimization can be further improved. ¡§While Taq is a standard product used in 90% of molecular biology labs today, scientists still must spend time optimizing their PCR reactions, which can be time-consuming and tedious,¡¨ she says. ¡§In the future, we should expect to see more products that simplify and speed up the PCR step so that researchers can focus on results.¡¨