Distinction of single base mismatches in duplex DNA using methylene blue as optical indicator.

A simple and sensitive approach for the recognition of single base mismatches in duplex DNA was developed. The single base mismatched double-strand (ds) DNA and the completely complementary dsDNA can quench the fluorescence of methylene blue to the different values, so the point mutation dsDNA can be identified from the duplex DNA effectively. The fluorescence intensity was decreased with the increasing dsDNA concentration in the range of 10~1000 nM, the detection limit was estimated to be 50 nM (S/N = 3). The contrast experiment showed that this method possesses the universality for the distinction of other single base mismatches in duplex DNA. Moreover, the interaction mechanism between methylene blue and dsDNA was also discussed in detail. The UV-Visible absorption and the fluorescence experiments indicated that the interaction of methylene blue and dsDNA had at least two effective modes: the electrostatic binding and the intercalation binding. When more methylene blue molecules bound onto mismatched dsDNA, the fluorescence intensity of methylene blue would decrease to a lower value than that of completely complementary dsDNA. Therefore, single base mismatched dsDNA can be recognized from duplex DNA effectively.