Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition

MutS protein is an important part of the DNA repair system which can specifically recognize and bind all possible single-base mismatches as well as 1-4 base insertion or deletion loops with varying affinities independent of other proteins or cofactors. In this paper, a new approach for electrochemical gene mutation detection based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated methylene blue (MB) markers for electrochemical signal generation is described. This method involves the immobilization of MutS protein onto the gold electrode, the hybridization of target DNA to form homoduplex or heteroduplex DNA, the application of MutS protein for the mutation recognition, and finally the intercalation of MB. The background is very low because MutS protein binds DNA containing mispaired and unpaired bases but does not bind equally well to DNA without mismatches or single-stranded DNA. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 5.6 x 10(-13)M, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.