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.
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页码：999 / 1005
页数：7
相关论文
共 17 条
[1]  
BOOSALIS MS, 1987, J BIOL CHEM, V262, P14689
[2]   Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides [J].
Fujiwara, T ;
Kimoto, M ;
Sugiyama, H ;
Hirao, I ;
Yokoyama, S .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2001, 11 (16) :2221-2223
[3]   BIOCHEMICAL BASIS OF DNA-REPLICATION FIDELITY [J].
GOODMAN, MF ;
CREIGHTON, S ;
BLOOM, LB ;
PETRUSKA, J .
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1993, 28 (02) :83-126
[4]   A unique unnatural base pair between a C analogue, pseudoisocytosine, and an A analogue, 6-methoxypurine, in replication [J].
Hirao, I ;
Kimoto, M ;
Yamakage, S ;
Ishikawa, M ;
Kikuchi, J ;
Yokoyama, S .
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2002, 12 (10) :1391-1393
[5]   Synthesis of 3-(2-deoxy-β-D-ribofuranosyl)pyridin-2-one and 2-amino-6-(N,N-dimethylamino)-9-(2-deoxy-β-D-ribofuranosyl)purine derivatives for an unnatural base pair [J].
Ishikawa, M ;
Hirao, I ;
Yokoyama, S .
TETRAHEDRON LETTERS, 2000, 41 (20) :3931-3934
[6]   Synthetically modified DNAs as substrates for polymerases [J].
Kool, ET .
CURRENT OPINION IN CHEMICAL BIOLOGY, 2000, 4 (06) :602-608
[7]   DNA replication fidelity [J].
Kunkel, TA ;
Bebenek, R .
ANNUAL REVIEW OF BIOCHEMISTRY, 2000, 69 :497-529
[8]   A specific partner for abasic damage in DNA [J].
Matray, TJ ;
Kool, ET .
NATURE, 1999, 399 (6737) :704-708
[9]   Efforts toward expansion of the genetic alphabet: DNA polymerase recognition of a highly stable, self-fairing hydrophobic base [J].
McMinn, DL ;
Ogawa, AK ;
Wu, YQ ;
Liu, JQ ;
Schultz, PG ;
Romesberg, FE .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1999, 121 (49) :11585-11586
[10]   An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo[(4,5)-b]pyridine [J].
Mitsui, T ;
Kitamura, A ;
Kimoto, M ;
To, T ;
Sato, A ;
Hirao, I ;
Yokoyama, S .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2003, 125 (18) :5298-5307
12