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Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) labeling of the cleavage products. The individual RCA amplicon molecules were counted on the surface using fluorescence microscopy. Two principal limitations of such single-molecule counting are the variability in the amplicon size, which results in a large variation in fluorescence signal intensity from the dye-labeled DNA molecules, and a high level of background fluorescence. It is shown here that an excellent alternative to RCA labeling is tagging with gold nanoparticles followed by imaging with a scanning electron microscope. Gold nanoparticles have a uniform diameter (15 +/- 0.5 nm) and provide excellent contrast against the background of the silicon substrate employed. Individual gold nanoparticles are readily counted using publicly available software. The results demonstrate that the labeling efficiency is improved by as much as approximately 15-fold, and the signal-to-noise ratio is improved by approximately 4-fold. Detection of individual cleaved DNA molecules following surface-invasive cleavage was linear and quantitative over 3 orders of magnitude in amount of target DNA (10(-18)-10(-15) mol).

Quantitative Detection of Individual Cleaved DNA Molecules on Surfaces Using Gold Nanoparticles and Scanning Electron Microscope Imaging