In this competitive renewal proposal we seek to continue the development of """"""""surface invader"""""""" DNA arrays for high throughput SNP analysis. During the prior grant period we developed the surface invader assay and demonstrated """"""""proof-of-principle"""""""" for the parallel analysis of SNPs directly from unamplified human genomic DNA samples on spotted DNA arrays. Now we desire to take this technology to the next step, i.e. to implement this chemistry in a high-density DNA array format, where the DNA array is manufactured by photolithographic methods. There are three issues that will be addressed to enable fabrication of the needed DNA arrays. First, chemistry will be implemented for the photolithographic synthesis of DNA molecules in the 5'->3' direction. Second, chemistry will be developed and implemented to allow the photolithographic synthesis of two different sequences intermixed in a single array element; both are needed to form the / necessary ternary complex on the surface. Third, glassy carbon and/or diamond thin film substrates will be developed for use in photolithographic DNA synthesis. A secondary thrust of this proposal will be to investigate an approach to detection based upon the detection of single molecule cleavage events on the surface. The rolling circle amplification reaction can make DNA molecules hundreds of thousands of bases long from a single initiation site. As it is very easy to detect such long DNA molecules, this raises the possibility of directly detecting individual cleaved DNA molecules on the surface; in fact, our preliminary results have shown this capability. This is intriguing, as it opens the possibility of analyzing very low levels of nucleic acid targets, perhaps as little as a single molecule, with many applications in areas such as gene expression analysis, infectious disease diagnostics, biowarfare agent detection, and genotyping from minute samples such as buccal swabs or fingerprick blood samples.
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