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Greater Amplification Specificity with New Hot Start PCR Enzyme



SureStart Taq DNA polymerase makes hot start simple and efficient

Jonathan Eads Frances Bai Ronda Allen Holly Hogrefe

Stratagene

SureStart Taq DNA polymerase is a modified form of Taq2000 DNA polymerase with hot start capability to increase PCR specificity and improve performance. Using SureStart Taq DNA polymerase, hot start is incorporated into PCR protocols previously optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions. SureStart Taq DNA polymerase can be used in a variety of amplification systems to improve sensitivity, specificity, and product yield.

Certain PCR enzymes exhibit significant polymerase activity at temperatures encountered during reaction setup or while ramping up to stringent primer annealing temperatures. For example, Taq DNA polymerase exhibits 2% maximum activity at 25C (room temperature) and 70% maximum activity at 50C (generally at or below the Tm of PCR primers).1 When PCR reactants are combined at temperatures below stringent primer annealing temperatures, nonspecific primer annealing and extension can occur. Once formed, misprimed products and primer oligomers are efficiently amplified throughout remaining PCR cycles. Undesired products and primer-dimers can impair gel analysis, quantitation, and sequencing of specific PCR products. Moreover, synthesis of misprimed products diverts reaction components (dNTPs, primers) away from the production of desired products, which leads to poor amplicon yield.

Various techniques have been developed to reduce nonspecific primer extension by Taq DNA polymerase at low temperatures. One method involves replacing Taq with Stratagenes Pfu DNA polymerase, which is less prone to zero-cycle artifacts due to lower activity at nonrestrictive temperatures (e.g., 8% maximum activity at 50C).1 Another method for reducing nonspecific amplification employs Stratagenes Perfect Match PCR enhancer, which decreases background by destabilizing mismatched primer-template complexes.2,3 In addition, various hot start procedures have been developed to prevent nonspecific primer extension at low temperatures. In these procedures, a critical PCR component (e.g., dNTPs, polymerase, or Mg2+) is withheld until stringent primer annealing temperatures are reached.4-7 The missing component can be added manually at the initial denaturation step4,5 or withheld by a physical wax barrier until temperatures sufficient to melt wax are achieved.6

More recently, reversibly inactivated DNA polymerases have been used to provide hot start. Heat-reversible inhibition is accomplished using neutralizing antibodies,8 oligonucleotide inhibitors,9 or enzyme modification.10 DNA polymerases reversibly inactivated by antibodies regain activity upon denaturation of the antibody (1 minute at 80C 8) during ramp-up to initial denaturation temperatures. In comparison, commercially available preparations of modified Taq remain inactive until higher temperatures are reached (>4 minutes at 95C 10). As with other hot start procedures, the use of reversibly inactivated DNA polymerases allows for PCR setup at room temperature, while providing reduced background and enhanced amplification of specific product. Moreover, hot start PCR enzymes can be introduced into existing amplification systems with little or no modification of PCR protocols, and their use avoids awkward manipulations associated with wax barrier or manual methods.

Eliminate Nonspecific PCR Products

SureStart Taq DNA polymerase is a chemically inactivated version of Stratagenes Taq2000 DNA polymerase1, a highly purified recombinant Taq DNA polymerase preparation. SureStart Taq DNA polymerase is provided in an inactive state and remains completely inactive until stringent denaturation temperatures are reached. The enzymes accompanying 10X reaction buffer has been formulated to provide flexible and efficient enzyme activation, resulting in optimal specificity and product yield. SureStart Taq DNA polymerase can be partially or completely activated in a pre-PCR heat step (9 to 12 minutes at 92 to 95C Pre-PCR Heat Activation Method) or it can be activated slowly during the denaturation steps of thermal cycling (PCR Heat Activation Method).

In Figure 1, results from the Pre-PCR and PCR Heat Activation Methods are compared. In this example, a 105-bp fragment of the glucocerebrosidase gene was amplified from human genomic DNA using Taq (Figure 1A) or SureStart Taq (Figure 1B) DNA polymerase. Taq DNA polymerase amplified numerous extraneous bands in addition to the desired product (Figure 1A, Lanes 1-5). In contrast, background was dramatically reduced in reactions carried out with SureStart Taq DNA polymerase (Figure 1B). High product yields were achieved after 30 to 33 cycles when SureStart Taq was activated prior to temperature cycling during a 10-minute incubation step (95C) added at the beginning of the thermal cycling program (Figure 1B, Lanes 1-5). When the pre-PCR heat step was omitted, 36 to 39 cycles were required to achieve comparable product yields (Figure 1B, Lanes 6-10). Although the PCR heat activation method generally requires more cycles (35 to 45 cycles) to achieve optimal product yield, greater amplification specificity may be realized due to lower enzyme concentrations in initial PCR cycles.

Superior PCR Performance

Amplification of low-copy-number templates is especially problematic with complex DNA samples due to frequent mispriming events at ambient temperature. Hot start methods eliminate mispriming and thereby improve the detection of low-copy-number targets. Challenging model amplification systems were used to evaluate the hot start properties of SureStart Taq DNA polymerase. In the systems employed in Figures 2 and 3, amplicons were synthesized from serially diluted DNA templates in the presence of high concentrations of denatured genomic DNA. In Figure 2, a 115-bp fragment of the gag gene was amplified from 5 to 5000 copies of HIV-1 DNA in the presence of 0.1 mg of denatured human genomic DNA. While Taq DNA polymerase successfully amplified the target from 500 and 5000 copies of HIV-1 template, nonspecific products predominated in reactions using 5, 50, and 500 copies (Figure 2). In contrast, SureStart Taq DNA polymerase produced specific amplification product from as few as five copies of DNA template, and no background was visible (Figure 2).

In Figure 3, a 970-bp fragment was amplified from 101 to 105 copies of lambda DNA in the absence or presence of 0.5 mg of denatured genomic DNA. In a complex DNA sample, multiple nonspecific products were generated, and specific product yield was reduced (Figure 3A). When compared to Taq, SureStart Taq DNA polymerase successfully amplified the desired product from as few as 101 copies of lambda DNA, even in the presence of high concentrations of extraneous denatured DNA. Moreover, SureStart Taq DNA polymerase generated far fewer nonspecific products compared to Taq DNA polymerase. Comparisons with other vendors hot start enzymes showed similar improvements in specific product yield (Figure 3B, Lanes 1 to 3) but high background was evident in two of the three hot start preparations tested.

Conclusions

Use Stratagenes SureStart Taq DNA polymerase to achieve a simple and effective method for hot start. SureStart Taq DNA polymerase improves the sensitivity of amplification reactions by reducing background and increasing yield of desired product. Compared to other vendors hot start Taq preparations, SureStart Taq provides comparable or superior detection of low-copy-number targets. SureStart Taq is inactive at ambient temperatures, facilitating room-temperature PCR setup and high-throughput PCR analyses. Moreover, high sensitivity and room-temperature assembly can be achieved with little or no changes to existing procedures. The benefits of SureStart Taq DNA polymerase in quantitative PCR and RT-PCR applications were highlighted in a previous article that discussed Stratagenes new Brilliant PCR and RT-PCR core reagent kits.12

Methods

PCR reactions were assembled at room temperature. PCR amplifications (50 ml) employed 200 mM each dNTP, 0.3 to 0.5 mM each primer, and the indicated amounts of DNA template (figure legends). Amplifications were carried out using 2.5 U of SureStart Taq or Taq2000 DNA polymerase (Stratagene) and the recommended PCR buffer. Enzyme comparisons with other vendors hot start Taq antibody or hot start Taq preparations were carried out under identical conditions, except that the recommended PCR buffer and activation method was used.

PCR reactions were amplified in a RoboCycler Gradient 96 temperature cycler (Stratagene) fitted with a Hot Top device and using 200-ml thin-walled PCR tubes. Enzyme comparisons employed identical temperature cycling parameters, except that a heat step consisting of 93C to 95C for 10 minutes was added to the beginning of each thermal cycling program for SureStart Taq reactions. The 105-bp target (Figure 1) was amplified using the following conditions: 1 cycle at 95C for 1 minute (Taq) or 10 minutes (SureStart Taq), followed by 30 to 42 cycles at 95C for 1 minute (denaturation); 54C for 1 minute (annealing); and 72C for 1 minute. The conditions used to amplify the 115-bp gag gene fragment (Figure 2) were 1 cycle at 95C for 1 minute (Taq and Taq plus hot start antibody) or 10 minutes (SureStart Taq), followed by 40 cycles at 95C for 1 minute (denaturation); 55C for 1 minute (annealing); and 72C for 1 minute, followed by one final extension cycle of 72C for 10 minutes. The conditions used to amplify the 970-bp lambda DNA fragment (Figure 3) were 1 cycle at 93C for 1 minute (Taq and Taq plus hot start antibody), 10 minutes (SureStart Taq) or 10 to 15 minutes (competitors modified Taq preparations); 45 cycles at 93C for 1 minute (denaturation); 60C for 50 seconds (annealing); and 68C for 1 minute, followed by one final extension cycle of 68C for 10 minutes.


REFERENCES

1. Nielson, K.B., Cline, J., and Hogrefe, H. (1997) Strategies 10: 40-43.
2. Nielson, K., et al. (1991) Strategies 4: 38.
3. Bai, F. and Hogrefe, H. (1999) Strategies 12: 14-16.
4. Mullis, K. (1991) PCR Methods Appl. 1: 1-4.
5. Aquila, R.T., et al. (1991) Nucl. Acids Res. 19: 3749.
6. Chou, Q., et al. (1991) Nucl. Acids Res. 20: 7.
7. Erlich, H.A., Gelfand, D., and Sninsky (1991) Science 252: 1643-1651.
8. Sharkey, D.J., et al. (1994) BioTechnology 12: 506-509.
9. Lin, Y. and Jayasena, S.D. (1997) J. Mol. Biol. 271: 100-111.
10. Birch, D.E., et al. (1996) Nature 381: 445-446.
11. Nielson, K.B., et al. (1996) Strategies 9: 7-8.
12. Padmabandu, G. et al (2000) Strategies 13: 88-92.


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