A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

Shi-Ying LI; Xiang-Yu ZHANG; Xin ZHANG; Yan LAN; Zi-Chun HUA

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Volume 23 Issue 6

Dec. 2010

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Article Navigation > Biomedical and Environmental Sciences > 2010 > 23(6): 496-501

Shi-Ying LI, Xiang-Yu ZHANG, Xin ZHANG, Yan LAN, Zi-Chun HUA. A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity[J]. Biomedical and Environmental Sciences, 2010, 23(6): 496-501.

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Shi-Ying LI, Xiang-Yu ZHANG, Xin ZHANG, Yan LAN, Zi-Chun HUA. A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity[J]. Biomedical and Environmental Sciences, 2010, 23(6): 496-501.

A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China;Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Jiangsu, China
The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China
The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China;Changzhou High-Tech Research Institute of Nanjing University and Changzhou Target Pharma Laboratories Inc., Changzhou 213164, Jiangsu, China

Funds: the Chinese National Natural Sciences Foundation(30821006%50973046)%the Doctoral Station Science Foundation from the Chinese Ministry of Education(200802840023)%the Jiangsu Provincial Natural Science Foundation(BK2010046%BK2008138%BK2008072%BY2009147)

Abstract

Objective The assay of DNA mismatch repair (MMR) activity can be used as a biomarker for environmental condition detection and human disease diagnosis. Radioactive 32P-endlabeled DNA containing mismatch is extensively used as the substrate for MMR activity analyses. The aim of the present study is to develop a simple non-radioactive, but equally specific and sensitive method for the MMR activity assay. Methods A fluorescent label was chosen to replace the radioactive isotope label. Sensitive evaluation of the fluorescent label was carried out for the first time, and then the fluorescent label was compared with the isotope label in the MMR activity and DNA binding assays. Result LOD (limit of detection) of the fluorescent label was about 0.1 fmol and the relative signal strength displayed a pretty good linear relationship. Moreover, the fluorescent label method has equivalent sensitivity and performance as compared with the classical radioactive method in experiments. Conclusion In light of the sensitivity, reproducibility, safety, rapidity and long lifespan of the fluorescent label,this improved method can be applied to evaluation of biologic and toxic effects of environmental pollutants on man and other forms of life.
- Fluorescent label,
- MMR activity,
- EMSA,
- TDG
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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China;Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Jiangsu, China

The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China

The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, School of Stomatology and Affiliated Stomatological Hospital, Nanjing University, Nanjing 210093, Jiangsu, China;Changzhou High-Tech Research Institute of Nanjing University and Changzhou Target Pharma Laboratories Inc., Changzhou 213164, Jiangsu, China

Key words:

Fluorescent label /
MMR activity /
EMSA /
TDG

Abstract: Objective The assay of DNA mismatch repair (MMR) activity can be used as a biomarker for environmental condition detection and human disease diagnosis. Radioactive 32P-endlabeled DNA containing mismatch is extensively used as the substrate for MMR activity analyses. The aim of the present study is to develop a simple non-radioactive, but equally specific and sensitive method for the MMR activity assay. Methods A fluorescent label was chosen to replace the radioactive isotope label. Sensitive evaluation of the fluorescent label was carried out for the first time, and then the fluorescent label was compared with the isotope label in the MMR activity and DNA binding assays. Result LOD (limit of detection) of the fluorescent label was about 0.1 fmol and the relative signal strength displayed a pretty good linear relationship. Moreover, the fluorescent label method has equivalent sensitivity and performance as compared with the classical radioactive method in experiments. Conclusion In light of the sensitivity, reproducibility, safety, rapidity and long lifespan of the fluorescent label,this improved method can be applied to evaluation of biologic and toxic effects of environmental pollutants on man and other forms of life.

Citation: