A surface invasive cleavage assay for highly parallel SNP analysis

The structure-specific invasive cleavage of single-stranded DNA by 5′ nucleases is a useful means for sensitive detection of single-nucleotide polymorphisms or SNPs. The solution-phase invasive cleavage reaction has sufficient sensitivity for direct detection of as few as 600 target molecules with no prior target amplification. One approach to the parallelization of SNP analysis is to adapt the invasive cleavage reaction to an addressed array format. Two surface invasive cleavage reaction strategies were designed and tested using the polymorphic site in codon 158 of the human ApoE gene as a model system, with a synthetic oligonucleotide as target. The upstream oligonucleotide, which is required for the invasive cleavage reaction, was either added in solution (strategy 1), or co-immobilized on the surface along with the probe oligonucleotide (strategy 2). Both strategies showed target-concentration and time-dependent amplification of signal. Parameters that govern the rate of the surface-invasive cleavage reactions are discussed.