( High-fidelity PCR of genomic targ...)Enhanced Amplification of Long Targets with PfuTurbo DNA,,,Polymerase

High-fidelity PCR of genomic targets up to 19 kb

Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase

Michael Borns Holly Hogrefe
Stratagene

Improve amplification of long targets with PfuTurbo DNA polymerase* by making simple modifications to PCR reaction conditions. In this update, we describe adjustments that allow complex, genomic targets to be amplified up to 19 kb in length.

Superior accuracy and PCR performance have made Stratagenes PfuTurbo DNA polymerase the enzyme of choice for nearly all PCR applications.^1-5 Although PfuTurbo DNA polymerase can amplify complex genomic targets up to 10 kb in length,¹ the highest product yields are routinely achieved with genomic targets up to 6 kb. To achieve higher yields of 6-kb to 10-kb products, extension times can be increased from 1 minute to 2 minutes per kb of target.¹

A complementary set of PCR reaction conditions has been developed to further improve amplification of long genomic targets with PfuTurbo DNA polymerase. We found that amplification of complex targets greater than 6 kb in length is limited by buffer components, rather than the robustness of PfuTurbo DNA polymerase. To enhance PCRs of long targets, we recommend increasing dNTP concentrations from 200 M to 500 M each and increasing PCR buffer concentration from 1X to 1.5X.

Enhanced Reaction Conditions

Figure 1 shows the effects of buffer concentration on amplifications of 17- and 19-kb genomic targets with PfuTurbo DNA polymerase. PCRs were unsuccessful using standard conditions (Figure 1, Lanes 3 and 4; 1X buffer). In contrast, reactions carried out in 1.5X PCR buffer produced high yields of both genomic targets (Figure 1, Lanes 5 an
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