Single-nucleotide polymorphisms (SNP) are single base changes that occur within a DNA sequence and are linked to several genetic diseases. In typical microarray assays, the mismatches caused by these SNPs are difficult to detect at low concentrations because they must compete with more tightly binding fully-complementary strands. If each hybridization event is analyzed individually, it may be possible to discriminate between SNPs and fully complementary strands by monitoring their dissociation kinetics. In this work, we have applied total internal reflection fluorescence microscopy to study the kinetics of DNA hybridization at the single-molecule level, and the influence of SNPs on rates of association and dissociation. Single-stranded DNA (ssDNA) probe molecules were immobilized on a glass slide and exposed to a solution containing a fluorescently-labeled target ssDNA. The target in solution was either a non-complementary strand to measure background fluorescence and non-specific interactions, a fully complementary strand, or a complementary strand containing a single-base mismatch. At low target concentrations, individual hybridization events can be detected by the appearance of fluorescence from the target DNA label. By measuring the duration of each hybridization event, the dissociation kinetics can be measured at equilibrium. Because the fraction of probe sites that are occupied is very small (&lt;0.01), there is no competition for probe strands and displacement by target strands with higher affinities. The association constant, Ka, can be determined by counting the population of hybridized target DNA versus solution concentration; together with the dissociation rate, koff , the rate of association can be determined, kon = Ka koff, so that a complete picture of the hybridization kinetics is available. Previous studies of SNP dissociation in free solution suggested that changes in Ka compared to a fully complementary strand were due solely to changes in koff. Single-molecules kinetics reveal that changes in Ka between the fully complementary and mismatch strands are a result of changes in both association and dissociation rate constants. Furthermore, changes in the association and dissociation rate constants were found to depend on the identity and location of the mismatch.

Single-molecule detection of single-nucleotide polymorphisms (SNPs) in DNA hybridization kinetics

251st National Meeting (2016)

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