A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer

被引：9
|
作者：
Kimoto, M
Yokoyama, S
Hirao, I
机构：
[1] RIKEN Genom Sci Ctr, Prot REs Grp, Tsurumi Ku, Yokohama, Kanagawa 2300045, Japan
[2] RIKEN Harima Inst SPring8, Sayo, Hyogo 6795148, Japan
[3] Univ Tokyo, Grad Sch Sci, Dept Biophys & Biochem, Bunkyo Ku, Tokyo 1130033, Japan
[4] Univ Tokyo, Adv Sci & Technol Res Ctr, Meguro Ku, Tokyo 1538904, Japan
来源：
BIOTECHNOLOGY LETTERS | 2004年 / 26卷 / 12期
关键词：
DNA polymerase; GeneScan; kinetic parameters; single-nucleotide insertion; unnatural base pairs;
D O I：
10.1023/B:BILE.0000030047.32578.82
中图分类号：
Q81 [生物工程学（生物技术）]; Q93 [微生物学];
学科分类号：
071005 ; 0836 ; 090102 ; 100705 ;
摘要：
A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5'-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication.
引用
收藏
页码：999 / 1005
页数：7
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