Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase.
DNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and betaG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG(1), <font face="k">k. K(d) values were 7.3 x 10(-8) for 3G8 and 1.1 x 10(-6) for betaG1. Nucleotide sequencing of cDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among alpha-like (family B) DNA polymerases (Region II), and the epitope for betaG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).
Mizuguchi
H
H
Department of Biotechnology, Graduate School of Engineering Osaka University, Suita, Osaka, 565-0871, Japan.
Nakatsuji
M
M
Fujiwara
S
S
Takagi
M
M
Imanaka
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000276:immunology* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D019711:Pyrococcus / Q000201:enzymology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase