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[Influence of DNA polymerases on mutation screening of denaturing high-performance liquid chromatography]
Man-ran Liu1, Kai-feng Pan, Yi Wang
1Beijing Institute for Cancer Research, Beijing Laboratory of Molecular Oncology, School of Oncology, Peking University, Beijing 100034, P. R. China.
Background & Objective:
Recently, a sensitive and accurate technique called denaturing high-performance liquid chromatography (DHPLC) has been used in gene mutation screening, but several factors, especially DNA polymerase were discovered to affect the resolution of DHPLC technique. In order to find one or more suitable polymerases for DHPLC analysis, we designed this study to evaluate the influence of non-proofreading and proofreading DNA polymerase on mutation screening with DHPLC.
Methods:
Three fragments which were 268 bp, 317 bp, and 590 bp in size were chosen for DHPLC analysis. To evaluate the negative influence of polymerase on DHPLC resolution, the same fragments were amplified by using 11 kinds of DNA polymerases [6 brands of Taq, 1 kind of Taq gold and, 4 sorts of proofreading DNA polymerase (Pfu and Vent)] and those PCR products were analyzed by DHPLC. The chromatographical profile were identified and compared.
Results:
There were two kinds of negative influences on DHPLC analysis chromatography profiles. There were no or tiny influences induced by proofreading or Taq gold polymerases.
Conclusions:
Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase. Especially, Pfu DNA ought to be preferentially selected to amplify GC-rich small fragments or other influence-prone fragments when mutation is detected by DHPLC analysis.