Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially availab le DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 45 04-4510]. However, when long distance PCR(>5 kbp) was performed with KOD DN A polymerase, amplification efficiency (product yield) becomes lower becaus e of its strong 3'-5' exonuclease activity for proof-reading. In order to i mprove a target length limitation in PCR, mutant DNA polymerases with decre ased 3'-5' exonuclease activity were designed by substituting amino acid re sidues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn 210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases we re altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase ( WT) and its exonuclease deficient mutant (N210D) were mixed in different ra tio and their characteristics in PCR were examined. When the mixed enzyme ( WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mut ation frequency could be efficiently achieved. (C) 2001 Elsevier Science B. V. All rights reserved.