( Improve PCR product yield and...)Optimizing pfuturbo DNA Polymerase Amplification Reactions with,,,Perfect Match PCR Enhancer

Improve PCR product yield and specificity for difficult amplifications

Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer

Frances Bai Holly Hogrefe
Stratagene

Certain PCR amplification systems produce multiple product bands or products of the wrong size due to nonspecific priming events, template secondary structures, or suboptimal PCR conditions. Stratagenes Perfect Match PCR enhancer *^, improves the yield and specificity of problematic PCR reactions carried out with Taq DNA polymerase. We investigated the effects of Perfect Match PCR enhancer on amplifications performed with pfuturbo DNA polymerase,**^, a robust high-fidelity PCR enzyme recently developed by Stratagene. Using PfuTurbo polymerase, the addition of the enhancer substantially improved the yield and specificity of suboptimal PCR systems; furthermore, PCR product yields and target-length capability were higher than for Taq plus Perfect Match enhancer reactions.

Stratagene is committed to developing innovative, high-performance PCR enzymes and PCR-enhancing agents to maximize product yield, specificity, and fidelity. Stratagene recently introduced PfuTurbo DNA polymerase, a special formulation of cloned Pfu DNA polymerase and a novel thermostable PCR-enhancing factor*** (the Turbo additive) that dramatically improves PCR product yields without altering DNA replication fidelity.¹ PfuTurbo DNA polymerase is ideally suited for amplifying genomic DNA targets up to 10 kb and cloned targets up to 15 kb with the greatest accuracy possible.¹ PCR comparisons have shown that PfuTurbo DNA polymerase amplifies longer targetsin higher yieldthan Pfu DNA polymerase (alone), Taq DNA polymerase, and other commercially available single-enzyme formulations.^1,2

Stratagenes PCR-enhancing Agents

Perfect Match PCR enhancer improves the yield and specificity of PCR amplification reactions carried out with Taq DNA polymerase.^3,4,5 Perfect Match enhancer reduces or eliminates nonspecific amplification, possibly by destabilizing mismatched primer-template complexes that can lead to false priming and the generation of undesired PCR artifacts. While Perfect Match enhancer improved amplifications from both plasmid and genomic DNA templates, these optimal results depended on the ratio of Perfect Match enhancer to DNA template employed.⁴ Additional studies revealed that Perfect Match enhancer also improves amplifications of longer genomic DNA targets (2 to 5 kb) using Taq DNA polymerase; presumably by reducing secondary structure formation in the template strand, which impedes translocation of the DNA polymerase and limits amplification of longer targets.⁵

Perfect Match PCR enhancer and the Turbo additive in the PfuTurbo DNA polymerase formulation are distinct factors, which mediate PCR enhancement via different mechanisms. It is therefore likely that using Perfect Match enhancer could further improve the performance of PfuTurbo DNA polymerase, as has been demonstrated previously for Taq DNA polymerase PCR amplifications.^3,4,5 Below we describe the effects of Perfect Match enhancer on amplifications of several difficult primer-template systems using PfuTurbo DNA polymerase.

Adding Perfect Match Enhancer to PCR Reactions

PCR amplifications were conducted with 5 U of PfuTurbo DNA polymerase or Taq2000 DNA in the enzymes recommended buffer, using 200 M each dNTP, 100 ng of genomic DNA, 100 ng of each oligonucleotide primer, and 1 U (1 l) of Perfect Match PCR enhancer per 50-l reaction. A 120-bp portion of the glucocerebrosidase gene was amplified from human genomic DNA and the 6.2- and 6.6-kb fragments were amplified from Big Blue transgenic mouse genomic DNA that contained 40 copies of lambda phage DNA per genome.

PCR reactions were conducted in a Stratagene RoboCycler Gradient 96 temperature cycler fitted with a Hot Top Assembly, using 200-l thin-walled PCR tubes. Temperature cycling parameters were devised for the 120-bp target: 1 cycle at 94 for 1 minute, followed by 30 cycles at 94C for 1 minute (denaturation); 54C for 1 minute (annealing) and 72C for 1 minute (extension); and 1 final extension cycle of 72C for 10 minutes. The amplified reaction products were electrophoresed on a 4 to 20% TBE/acrylamide gel, stained with ethidium bromide, and imaged using the Eagle Eye II still video system. Temperature cycling parameters were established for the 6.2-kb target: 1 cycle at 95C for 1 minute, followed by 30 cycles at 95C for 1 minute (denaturation); 58C for 1 minute (annealing) and 72C for 7 minutes (extension); and 1 final extension cycle of 72C for 10 minutes. The same cycling conditions were used for the 6.6-kb target, except we employed a 60C annealing temperature. For the long targets, the amplified reaction products were electrophoresed on a 1% agarose/1X TBE gel.

Difficult PCR Amplification Systems

Figure 1

To demonstrate the variety of improvements Perfect Match PCR enhancer can have on PCR performance, we selected primer-template systems which generate multiple bands (Figure 1 and Figure 3) or bands of the wrong size (Figure 2).

Figure 2

Improved Product Yield and Specificity

The first system tested was designed to amplify a 6.2-kb fragment from mouse genomic DNA. In the absence of Perfect Match enhancer, PfuTurbo DNA polymerase synthesized multiple products, including the desired 6.2-kb band, while Taq DNA polymerase generated a smear (Figure 1). However, adding Perfect Match enhancer dramatically increased the yield of desired product in both the PfuTurbo polymerase and Taq amplification reactions (Figure 1). In the PfuTurbo polymerase plus Perfect Match enhancer reaction, undesired side products were still produced, but the relative proportions of each product were altered such that the percentage of desired product (6.2 kb) dramatically increased. Perfect Match enhancer was required for obtaining the Taq generated product, and it also greatly reduced smearing. The lower product yield achieved with Taq DNA polymerase is consistent with Taqs reduced target-length capability complared to PfuTurbo DNA polymerase.

Successful Amplification of Long Targets

The second amplification system tested (Figure 2) was designed to amplify a 6.6-kb fragment from the same template described in Figure 1. In the absence of Perfect Match enhancer, PfuTurbo DNA polymerase synthesized a small amount of a shorter undesired product, while Taq DNA polymerase produced a smear. In the presence of Perfect Match enhancer, both enzymes produced the specific product but product yield was higher for the PfuTurbo amplification.In cases where Perfect Match enhancer must be present to amplify relatively long targets (e.g., Figure 1 and Figure 2), it may act by reducing secondary structure formation in the template strand, which impedes translocation of the DNA polymerase.⁵

Improved Specificity

Figure 3 shows data using a third, unrelated amplification system designed to synthesize a 120-bp fragment from human genomic DNA. In the absence of Perfect Match enhancer, both PfuTurbo DNA polymerase and Taq DNA polymerase produce multiple undesired side products. Adding Perfect Match enhancer significantly decreases the synthesis of larger, undesired side products by both enzymes. Unlike amplifications of long complex targets (Figure 1 and Figure 2), PfuTurbo and Taq DNA polymerases synthesized similiar yields of the relatively short amplicon.

Conclusions

Perfect Match enhancer provides a quick, convenient solution for reducing amplification of undesired PCR side products. Use Perfect Match PCR enhancer to improve the yield and specificity of difficult PCR amplification systems employing both PfuTurbo DNA polymerase and Taq DNA polymerase. The distinct PCR-enhancing activities of Perfect Match enhancer and the Turbo additive work in concert with Pfu DNA polymerase to provide high product yields with minimal PCR artifacts and replication errors. Perfect Match PCR enhancer may be purchased separately or as part of Stratagenes Opti-Prime PCR optimization kit. This kit consists of a set of buffers and PCR-enhancing agents (including formamide, DMSO, and Perfect Match PCR enhancer) for use in an easy, two-step protocol to optimize difficult PCR amplification reactions.⁶

REFERENCES

1. Hogrefe, H. et al. (1997) Strategies 10: 93-96.

2. Hogrefe, H., Bai, F., and Cline, J. (1998) Strategies 11: 36-37.

3. Nielson, K. and Mathur, E.J. (1990) Strategies 3: 17-22.

4. Lundberg, K.S., Mathur, E.J. (1991) Strategies 4: 4-5.

5. Nielson, K.B., Wilbanks, A., Hansen, C., and Mathur, E.J. (1991) Strategies 4: 38.

6. Schoettlin, W., Nielson, K.B., and Mathur, E. (1993) Strategies 6: 43-44.

* U.S. Patent Nos. 5,773,257, 5,605,824, 5,646,019 and patents pending
** U.S. Patent No. 5,545,552 and patents pending
*** Patents pending