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 extensive...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.展开更多
基金grants from 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)
文摘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.
