High Fidelity, Long and GC Rich PCR
The PCR accuracy achievable with Taq DNA Polymerase (~one error per 45,000 nucleotides incorporated) is not always sufficient. For demanding cloning, sequencing and expression applications, polymerases with proofreading activity (3’→5’ exonuclease activity) are preferred. These proofreading enzymes can reduce PCR error rates by 10 fold or more, depending on the polymerase.
Pfu DNA Polymerase, originally isolated from Pyrococcus furiosus, is one of the most commonly used proofreading enzymes. The unmodified form exhibits approximately 8–10 times higher accuracy than Taq DNA Polymerase. PCR fidelity, however, depends greatly on reaction conditions, including pH, magnesium concentration, balance of dNTPs, base composition of the template and purity of the components.
When working with Pfu DNA Polymerase, be aware that its 3’→5’ exonuclease activity might cause primer degradation. Generally, primers for use with the Pfu enzyme should be slightly longer than those for Taq, and the enzyme should always be the last reagent added to the reaction mixture. Pfu is slower than Taq and elongation of 1 kb requires 2 minutes, whereas Taq DNA Polymerase typically requires 1 minute.
The synergy of Taq DNA Polymerase and a proofreading DNA polymerase is used to generate longer PCR products with higher accuracy than that of Taq alone. Such enzymes blends can be used for the amplification of templates of up to 30–40 kb in length and of GC-rich templates. In the latter case, different additives (such as DMSO, formamide or betaine) are helpful to enhance the denaturation of GC-rich templates for higher yields.
High Fidelity and Long Range PCR Selection Guide
|GC rich PCR||Simplified Set-up||Optimization freedom||Blunt Cloning||High Yield|
|Pfu DNA Polymerase, 2.5 u/µl||Ten times more accurate than Taq DNA Polymerase; supplied with PCR Helper for better results||~10x higher than Taq||5 kb||+||+|
|Pfu PCR Master Mix, 2x||Includes Pfu DNA Polymerase and all other necessary High Fidelity PCR reagents||~10x higher than Taq||3 kb||+||+|
|Long & High Fidelity PCR Enzyme Mix, 2.5 u/µl
||A blend of thermostable DNA Polymerases for Long and High Fidelity PCR, supplied with PCR Helper for better results in GC PCR||Similar to Pfu||>30 kb||+||+||requires blunting||+|
|Long Range PCR Master Mix, 2x||Includes a blend of thermostable DNA Polymerases and all other necessary reagents for Long and High Fidelity PCR as well as for GC rich PCR||Similar to Pfu||>30 kb||+||+||requires blunting|
NOTE: All above enzymes and mixes are not recommended to be used in PCR with dUTP, as the proofreading polymerases bind to DNA templates with uracil and hypoxanthine what stalls DNA synthesis.