High-fidelity PCR of genomic targets up to 19 kb
Michael Borns Holly Hogrefe
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.
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 d 6). Increased performance in 1.5X buffer was partly, but not entirely, due to the 50% increase in MgSO4 concentration (data not shown). PCR buffer titration experiments, carried out with additional PCR systems, indicate that a PCR buffer concentration of 1.5X is consistently optimal for long PCRs with PfuTurbo DNA polymerase.5
The error rate of PfuTurbo DNA polymerase in 1.5X PCR buffer was measured as described.5 No significant differences in error rates were observed when PCRs were performed in 1.5X PCR reaction buffer, compared to the 1X concentration (Table 1).
PCR Enzyme Error Rates
The error rate of PfuTurbo DNA polymerase in 1.5X PCR buffer was measured as described.5 No significant difference in error rates were observed when PCRs were performed in PCR reaction buffer at 1.5X, compared to the 1X concentration.
PCR reaction buffer
PfuTurbo DNA Polymerase
1X cloned Pfu buffer
PfuTurbo DNA Polymerase
1.5X cloned Pfu buffer
Taq DNA Polymerase
1X Taq buffer
11 .7 0.8
Follow the simple modifications described here, and nearly double the range of complex targets amplified by PfuTurbo DNA polymerase. Enhanced target-length capability further extends the utility of PfuTurbo DNA polymerase in demanding PCR applications that require the highest accuracy possible.
Hogrefe, H., et al. (1997) Strategies 10: 93-96.
Hogrefe, H., Bai, F., and Cline, J. (1998) Strategies 11: 36-37.
Braman, J. and Hogrefe, H. (1998) Strategies 11: 86-87.
Borns, M., et al. (1999) Strategies 12: 40-44.
* U.S. Patent Nos. 5,545,552, 5,866,395, and 5,948,663 and patents pending