DUKEHEALTH.ORG
SCHOOL OF MEDICINE
DUKE UNIVERSITY
High Resolution Melting (HRM) Analysis for SNP
and novel Mutation Detection
The Duke University DNA analysis facility has a Applied Biosystems 7500 Fast Real-Time PCR system for HRM analysis. HRM analysis is a low-cost, small-scale method for identifying genetic variation such as SNP’s and mutations.
Simple and fast, this method is based on PCR melting (dissociation) curve techniques and is enabled by the recent availability of improved double-stranded DNA (dsDNA)–binding dyes along with next-generation real-time PCR instrumentation and analysis software. HRM analysis can discriminate DNA sequences based on their composition, length, GC content, or methylation state.
“HRM analysis differs from standard melt curve analysis in three ways:
1. Chemistry—HRM analysis uses brighter dyes at higher concentrations.
2. Instruments—HRM analysis requires instruments that collect fluorescence data at finer temperature resolution.
3. Software—HRM analysis requires more sophisticated software which uses new fluorescent scaling algorithms and plots HRM analysis starts with PCR amplification of the region of interest in the presence of a dsDNA binding dye. This binding dye has a high fluorescence when bound to dsDNA and low fluorescence in the unbound state. Amplification is followed by a high resolution melting step using instrumentation capable of capturing a large number of fluorescent data points per change in temperature, with high precision. When the dsDNA dissociates (or melts) into single strands, the dye is released, causing a change in fluorescence. The result is a melt curve profile characteristic of the amplicon. HRM analysis requires no manual post-PCR processing, is performed in a closed-tube system, and has a low reaction cost relative to other methods used to study genetic variation.” Applied Biosystems
For more information and cost estimates, please contact the DNA Analysis Facility.