Optimizing cycling conditions
Optimizing PCR additives
Optimizing 3' to 5' exonuclease activity
Taq DNA Polymerase introduces more errors into the PCR product while copying the template than do so-called proofreading DNA polymerases. Once a mismatch occurs during synthesis, Taq DNA polymerase will either extend the mismatched strand or fall off the template strand, leading to mutated or incomplete PCR products, respectively. Although this does not generally affect PCR efficiency when amplifying shorter PCR fragments, amplification of longer PCR products can be significantly impaired by mismatches introduced during DNA synthesis.
Proofreading DNA polymerases contain an inherent 3' to 5' exonuclease activity that removes base-pair mismatches. Adding a small amount of a proofreading DNA polymerase to the PCR mixture therefore significantly improves the amplification efficiency of longer PCR products.