Mutation detection by stacking hybridization on genosensor arrays

A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled “stacking oligonucleotide.” and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the “capture probe” and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3’t-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivalized glass. “Operating parameters” examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1 Optimal mismatch discrimination with 9-mer probes occurs at 45‡C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45‡C. 2. At 25‡C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5′-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied.