ANALYSIS OF OLIGONUCLEOTIDE PROBE AFFINITIES USING SURFACE-PLASMON RESONANCE - A MEANS FOR MUTATIONAL SCANNING

A novel strategy for real-time analysis of oligonucleotide probe hybri dization based on detection by surface plasmon resonance is described. The design of the analysis, exploiting the rapid dissociation kinetic s of short oligonucleotides from their hybridization templates, allows monitoring in genuine sensor mode of equilibrium hybridization respon ses, circumventing the need for regeneration between sample cycles. Ap plied to a model system comprising oligonucleotide probes and differen t immobilized hybridization targets the effects of temperature, probe length, and nucleotide substitutions in template were investigated. Th e procedure described was observed to have an efficient discriminatory power with respect to end-mismatch situations. Affinity determination s of octamer probes showed good correlation between calculated T-m-val ues and probe affinities. From affinity data collected at different te mperatures thermodynamic parameters were determined, which correlated well with data obtained from theoretical calculations. The technique, modified to a simplified form, allowed detection of single nucleotide substitutions in a target template, suggesting that procedures for con firmatory DNA sequencing can be envisioned. (C) 1997 Academic Press.