Electrochemical detection of single-nucleotide mismatches: application of M-DNA.

The detection of a single-nucleotide mismatch in unlabeled duplex DNA by electrochemical methods is presented. Impedance spectroscopy is used to characterize a perfect duplex monolayer and three DNA monolayers differing in the position of the mismatch. The monolayers were studied as B-DNA (normal duplex DNA) and after conversion to M-DNA (a metalated duplex). Modeling of the impedance data to an equivalent circuit provides parameters that are useful in discriminating the four monolayer configurations. The resistance to charge transfer, R(CT), was lower for all duplexes after conversion to M-DNA. Contrary to expectations, R(CT) was also found to decrease for duplexes containing a mismatch. However, R(CT) was found to be diagnostic for mismatch detection. In particular, the difference in R(CT) between B- and M-DNA (deltaR(CT)) decreased from 190(22) omega.cm(2) for a perfectly matched duplex to 95(20), 30(20), and 85(20) omega.cm(2) for a mismatch at the top (distal), middle, and bottom (proximal) positions of the monolayer with respect to the gold surface. Further, a method to form loosely packed single-stranded (ss)-DNA monolayers by duplex dehybridization that is able to rehybridize to target strands is presented. Rehybridization efficiencies were in the range of 40-70%. Under incomplete hybridization conditions, the R(CT) was the same for matched and mismatched duplexes under B-DNA conditions. However, deltaR(CT) between B- and M-DNA, under incomplete hybridization, still provided a distinction. The deltaR(CT) for a perfect duplex was 76(12) omega.cm(2), whereas a mismatch in the middle of the sequence yielded a deltaR(CT) value of 30(15) omega.cm(2). The detection limit was measured and the impedance methodology reliably detected single DNA base pair mismatches at concentrations as low as 100 pM.