Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence.

We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are approximately 1fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.

Effect of solvent diffusion on the apomyoglobin-water interface.

Few techniques can identify interactions between proteins and individual water molecules when the protein is in solution. The present work has sought to bridge the gap between the molecular level studies and the search for a physical property of the solution (bathing the proteins) that would regulate the protein hydration level. The properties of the solution were varied by adding nondenaturing solutes and solvents to the protein solutions and then studying their effect on the intrinsic fluorescence of apomyoglobin. The resolution of the tryptophan emission into the two component spectra corresponding to tryptophans W7 (accessible to the solvent) and W14 (buried in the protein matrix) has allowed us to probe two specific parts of the protein. Whereas W14 is not affected when the medium is altered, the analysis of W7 fluorescence has shown that cosolvent diffusion plays a dominant role in the mobility of water molecules near the protein surface.