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Updated: Aug 7, 2026

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
Published on: August 6, 2012
A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using
Michiko Kimoto1, Shigeyuki Yokoyama, Ichiro Hirao
1Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Abstract:
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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