High-resolution melting analysis of single nucleotide polymorphisms

The technology of Single Nucleotide Polymorphism (SNP) detection has evolved steadily through mobility shift studies, mass cleavage product evaluations, heterodimer differences in chemical, conformational, and enzymatic properties, mass spectroscopy and sequencing, to allele-specific hybridization probe methods. Each method presented challenges of labor intensity, unreliable efficiencies, complicated optimizations, and issues of sample quantity and quality. Concurrently the value of SNP detection in basic research and personalized medicine has continued to grow. Accessing the secrets of genetic individuality is the next frontier in moving medicine from the description of very low frequency and highly deleterious nucleotide changes to the study of very low frequency polymorphisms, lower penetrance polymorphisms, and polymorphisms with public health importance. High-Resolution Melting (HRM) analysis of SNP status became an option for high throughput settings with the development of double-stranded dyes that do not interfere with PCR amplification in saturation, eliminate dye jumping, and nearest neighbor sequence changes influence melt temperature via amplicon strand locking chemistry. This method is able to distinguish transitions, transversions, and identify novel changes at or near the SNP of interest rapidly, inexpensively, and without post-amplification assay techniques or extensive technical interpretation of data. For probe or solid matrix based assays, the investigator initially defines a set of target sequences for binding. These assays are not only difficult due to the optimization of binding conditions but are unable to detect sequences that were not included in the design, often have marginalized binding due to a “one size fits all” reaction, and are not distinct in the case of heterozygotes.