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Real-time optical detection of competitive surface hybridization on microarrays

Application of microarrays for single nucleotide polymorphims (SNPs) has a limited appeal currently due to low reliability of experimental results. Theoretical and experimental studies of surface hybridization of heterozygous samples allow us to identify two factors of observed instabilities. First, reactions may not reach thermodynamic equilibrium in the course of the experiment and second, competitive displacement of low affinity species by high affinity species is the mechanism defining specificity of molecular recognition. Here we describe a real time optical detection arrangement that facilitated the detection of competitive displacement between a wild-type target and a SNP target. Results show that even when the SNP is an order of magnitude lower in concentration (100 pM) then the wild-type target, the kinetics of the SNP hybridization affects hybridization of the wild-type target. Additionally, results show that observed binding kinetics can be altered by adjusting the concentration of the SNP without changing the concentration of the wild-type target. These results have significance when considering what needs to be accounted for when analyzing real time hybridization data.