SPR study of DNA hybridization with DNA and PNA probes under stringent conditions.

Surface plasmon resonance (SPR) spectroscopy has been used for studying on-chip DNA hybridization to a PNA probe and its counterpart DNA probe of a 22-mer sequence. Two stringency control strategies are used for single base mismatch discrimination, namely (1) adding a denaturant, i.e. formamide (FA), into hybridization buffer and (2) coupling negative potentials for selective dehybridization of mismatch DNA. These two strategies have either not been used before or been less-well studied in SPR detection. An end-point SPR measurement protocol (no real-time hybridization profile recorded) is developed for detecting DNA hybridization in the presence of FA, to circumvent the problem that the refractive index of FA is out of the detectable range of the SPR equipment. The missing of real-time measurement of hybridization profile is compensated with QCM measurement. Under optimal conditions, i.e. 10mM PBS with 30% FA and 1mM PBS with 50% FA, single base mismatch DNA is detected with 1.7 and 2.8 times less hybridization signals compared with the perfect match DNA, with the DNA probe and PNA probe, respectively. Under negative potential of -0.2 to -0.4V (vs. Ag/AgCl), mismatch DNA dissociates more than perfect match DNA by 1.7-2.5 times from the DNA probe and 2.1-3.5 times from the PNA probe. The higher mismatch discrimination efficiency of the PNA probe under stringent conditions would be attributable to its higher intrinsic sequence selectivity.