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Improve Amplification Specificity with Hot Start PCR Enzyme


SureStart Taq DNA polymerase utilizes ultra-pure Taq2000 DNA polymerase

SureStart Taq DNA polymerase features a modified version of Taq2000 DNA polymerase with hot start capability. Using SureStart Taq, hot start is in- corporated into PCR protocols previously optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions. SureStart Taq can be used in a variety of amplification systems to improve specificity, yield, and amplification of difficult targets.

A 105 bp fragment of the glucocerebrosidase gene was amplified from human genomic DNA using SureStart Taq (lane 1), unmodified Taq (lane 2), a competitors antibody-based hot start Taq (lane 3), or a competitors modified Taq (lane 4) DNA polymerase preparation. PCR reactions were assembled at room temperature and employed 2.5 U of each enzyme and the recommended buffer. Identical temperature cycling parameters were used, except that a heat step 95C, 10 minute heat step was added to the beginning of the thermal cycling program for lanes 1 and 4.

Combining PCR reactants at low temperatures can lead to high background and low product yields in some amplification systems. Certain PCR enzymes exhibit significant polymerase activity at temperatures encountered during reaction set-up (4 to 25C) or while ramping up to stringent primer annealing temperatures. For example, Taq DNA polymerase exhibits 2 to 3% maximum activity at 25C (room temperature PCR set up) and 70% maximum activity at 50C (generally at or below the Tm of PCR primers)1. Non-specific primer annea ling and extension at non-restrictive temperatures produces undesirable products that are amplified throughout the remaining PCR cycles. Misprimed products and artifacts such as primer-dimers can impair gel analysis, quantitation, and sequencing of specific PCR products. Moreover, in the amplification of misprimed products, dNTPs and primers are diverted away from the synthesis of specific products, reducing overall yields.

Stratagenes SureStart Taq DNA polymerase improves amplification reactions by reducing background and increasing yield of desired product.

SureStart Taq DNA polymerase consists of a reversibly inactivated version of Stratagenes Taq2000 DNA polymerase, a highly purified recombinant Taq DNA polymerase preparation. SureStart Taq polymerase remains inactive until stringent denaturation temperatures are reached. It is activated by adding a heat step (9 to 12 minutes at 92 to 95C) to the beginning of thermal cycling programs (pre-PCR heat-activation method). Alternatively, the pre-PCR heat-activation step can be omitted to permit enzyme activation during temperature cycling. When the PCR heat-activation method is employed, it is generally necessary to add additional cycles to existing cycling programs to achieve optimal product yield.

SureStart Taq DNA polymerase provides superior or equivalent improvements in specificity and product yield compared to other vendors hot start Taq preparations. In the amplification system shown in the figure, Taq DNA polymerase synthesizes multiple extraneous bands in addition to the desired 105 bp fragment (lane 2). In contrast, background is eliminated in reactions employing SureStart Taq DNA polymerase (lane 1). SureStart Taq DNA polymerase provided improved specificity and higher product yield compared to other vendors antibody-based (lane 3) and modified (lane 4) Taq hot start formulations. Moreover, using SureStart Taq DN A polymerase, superior performance and the convenience of room temperature PCR setup was easily achieved with only minor changes to the temperature cycling program.

REFERENCES

  1. Nielson, K.B., Cline, J., and Hogrefe, H. (1997) Strategies 10: 40-43.


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