Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase beta.
In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase beta (pol beta) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent(R) (exo-), DNA polymerase IIIalpha and the Klenow fragment, and the mammalian polymerases DNA polymerase alpha and human DNA polymerase delta, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol beta. 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 beta. We have demonstrated for the first time that mammalian pol beta can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol beta is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.
Hirano
Ken
K
Nano-Bioanalysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Takamatsu, Kagawa 761-0395, Japan. hirano-ken@aist.go.jp
Yoshida
Yuichiro
Y
Ishido
Tomomi
T
Wada
Yukihisa
Y
Moriya
Naoji
N
Yamazaki
Naoshi
N
Mizushina
Yoshiyuki
Y
Baba
Yoshinobu
Y
Ishikawa
Mitsuru
M
D000818:Animals; D004253:DNA Nucleotidylexotransferase / Q000737:chemistry / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats
D015342:DNA Probes; D005456:Fluorescent Dyes; D009711:Nucleotides; D004253:DNA Nucleotidylexotransferase; D004258:DNA Polymerase III; D019951:DNA Polymerase beta; D019793:Fluorescein