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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease
Pintao Li1, Qiuyuan Lin1, Huiwen Xiong1
1Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai 200433, P. R. China.
Abstract:
In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.
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