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Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of
B C Ye1, K Ikebukuro, I Karube
1Research Institute of Biochemistry, Key State Laboratory of Bioreactor Engineering,ast China University of Science and Technology, Meilong Road 130, 200237 Shanghai, China.
Abstract:
The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented. A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template. The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase. The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume. This system was also applied to the determination of E.coli O157:H7.