Characterization and Application to Hot Start PCR of Neutralizing Monoclonal Antibodies against KOD DNA Polymerase.
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概要
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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), 3GS and βG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG1, κ. Kd values were 7.3×10-8for 3G8 and 1.1×10-6 for βG1. Nucleotide sequencing of eDNAs 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 a-like (family B) DNA polymerases (Region II), and the epitope for βG1 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).
- 社団法人 日本生化学会の論文
著者
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Nakatsuji Miki
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Takagi Masahiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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Fujiwara Shinsuke
Department Of Bioscience Graduate School Of Science And Technology Kwansei-gakuin University:researc
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Mizuguchi Hiroshi
Department of Biotechnology, Graduate School of Engineering Osaka University
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Imanakat Tadayuki
Departnnent of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University
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