The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of Single Nucleotide Polymorphisms (SNPs) directly from genomic DNA without need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potential large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed, and agreed well with experimental results.; Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces using similar surface attachment chemistry. Our work showed that the detection sensitivity is improved by a factor of approximately 100 (100 amole of DNA target compared to 10 fmole in the earlier work) by replacing the DNA-modified gold surface with a more stable DNA-modified diamond surface.; A new probe design based on the molecular beacon has been developed to further improve the detection sensitivity of invasive cleavage reactions for both solution- and solid-phase formats. The feasibility of using the molecular beacon probes in the invasive cleavage reaction was demonstrated. Comparisons between the conventional FRET probe and the molecular beacon probes in the reaction showed a better signal generation from the molecular beacon probes. Criteria regarding systematic design of the molecular beacon probes for the invasive cleavage reaction were discussed, and future applications of these newly designed probes are proposed.
展开▼
