Certain mixtures of proofreading and nonproofreading DNA polymerases have been shown to significantly increase PCR product yield and enhance amplification of longer targets compared to single-enzyme formulations.16-18 The enhanced performance of polymerase mixtures is presumably due to the capacity of one enzyme to complement the inability of a second enzyme to extend a primer through potential template obstructions. These potential obstructions include mispaired bases that cause nonproofreading polymerases to stall prematurely and disassociate from the primer-template,16 abasic gaps that cannot be bridged by polymerases lacking terminal transferase activity19 and template secondary structures20 such as GC-rich hairpins.
The TaqPlus Long PCR system was developed for PCR amplifications of long DNA target regions (up to 35 kb) using relatively short extension times (0.5-1 minute per kb target). The system includes the TaqPlus Long DNA polymerase mixture, consisting of Taq2000 DNA polymerase and cloned Pfu DNA polymerases, and two reaction buffers. The low-salt buffer is recommended for relatively short target (010 kb) amplifications, while the high-salt buffer is optimal for longer target (535 kb) amplifications. The TaqPlus Long PCR system typically generates higher yields of both short and long PCR products than can be obtained using either component enzyme alone.21 Including Taq2000 DNA polymerase in the TaqPlus Long polymerase formulation ensures that artifactual background smearing is minimal when long extension times (8 minutes) are used.