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.sub.CT], was lower for all duplexes after conversion to M-DNA. Contrary to expectations, [R.sub.CT] was also found to decrease for duplexes containing a mismatch. However, [R.sub.CT] was found to be diagnostic for mismatch detection. In particular, the difference in [R.sub.CT] between B- and M-DNA ([DELTA][R.sub.CT]) decreased from 190(22) [OMEGA]x[cm.sup.2] for a perfectly matched duplex to 95(20), 30(20), and 85(20) [OMEGA]x[cm.sup.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.sub.CT] was the same for matched and mismatched duplexes under B-DNA conditions. However, [DELTA][R.sub.CT] between B- and M-DNA, under incomplete hybridization, still provided a distinction. The [DELTA][R.sub.CT] for a perfect duplex was 76(12) [OMEGA]x[cm.sup.2], whereas a mismatch in the middle of the sequence yielded a [DELTA][R.sub.CT] value of 30(15) [OMEGA]x[cm.sup.2]. The detection limit was measured and the impedance methodology reliably detected single DNA base pair mismatches at concentrations as low as 100 pM.