High Resolution Melting Analysis
John F. Mackay and Carl T. Wittwer
from: PCR Troubleshooting and Optimization: The Essential Guide (Edited by: Suzanne Kennedy and Nick Oswald). Caister Academic Press, U.K. (2011)
Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA)- is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation. This chapter covers some of these developments and provides a guide to those wishing to establish this technique, as well as troubleshooting advice for those already underway read more ...