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Biosens Bioelectron. 2011 Jan 15;26(5):2699-702. doi: 10.1016/j.bios.2010.08.073. Epub 2010 Sep 9.

Rapid and sensitive detection of DNA polymerase fidelity by singly labeled smart fluorescent probes.

Author information

1
Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

Abstract

We report here a novel approach to monitor the DNA polymerase fidelity in detailed steps, including mispair extension, mispair formation and 3'→5' proofreading. The method is based on the photo-induced electron transfer between the natural base guanine and the labeled fluorophore. The G:T mispair extension catalyzed by the exonuclease-deficient Klenow fragment DNA polymerase (KF exo(-)) was easily detected and the effect of the nearest neighbor base pair on the mispair extension rate was clearly observed. More importantly, kinetics of the G:T, G:A and G:G mispair formation and extension under single turnover conditions were measured by continuous fluorescence-based assay for the first time. The probes also showed their applicability to discriminate the 3'→5' proofreading activity of different exonuclease-proficient DNA polymerases. The presented method may greatly simplify the screening and characterization procedures of the increasing number of polymerases that are thought to be potential targets for drug design and cancer treatment. It will also provide important information for deep understanding of the polymerase fidelity mechanism.

PMID:
20875730
DOI:
10.1016/j.bios.2010.08.073
[Indexed for MEDLINE]

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