Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

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• 作者：Ken Hirano ; Yuichiro Yoshida ; Tomomi Ishido ; Yukihisa Wada ; Naoji Moriya ; Naoshi Yamazaki ; Yoshiyuki Mizushina ; Yoshinobu Baba ; Mitsuru Ishikawa
• 关键词：High-density labeling ; DNA probe ; Mammalian DNA polymerase ; Fluorophore-labeled nucleotide analogue ; Consecutive incorporation ; Incorporation fidelity ; Single-molecule sequencing
• 刊名：Analytical Biochemistry
• 出版年：2010
• 出版时间：15 October 2010
• 年：2010
• 卷：405
• 期：2
• 页码：160-167
• 全文大小：1014 K

文摘

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase β (pol β) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent_R (exo-), DNA polymerase IIIα and the Klenow fragment, and the mammalian polymerases DNA polymerase α and human DNA polymerase δ, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol β. The kinetic parameters K_m and k_cat were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol β. We have demonstrated for the first time that mammalian pol β can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol β is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.