A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer.
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.
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Michiko
M
Kimoto
Shigeyuki
S
Yokoyama
Ichiro
I
Hirao
D000465:Algorithms; D020137:Base Pair Mismatch / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004261:DNA Replication / Q000235:genetics*; D019544:Equipment Failure Analysis; D007700:Kinetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D009711:Nucleotides / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D012371:Robotics / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D012984:Software; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
D009711:Nucleotides