High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis. The technique relies on quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and in combination with qPCR permits the identification of genetic variation in nucleic acid sequences by the controlled melting of a double-stranded PCR amplicon. As opposed to standard melt curves which are run for routing qPCR experiments, HRM melt curves involve the collection of melt data in 0.2 degree C increments. Furthermore, in order to identify small nucleotide changes it is essential to eliminate background fluorescence from any HRM analysis. Recent advances in real time PCR equipment, software and reagents (including fluorescent dyes) has turned HRM into a robust analytical technique capable of detecting a small proportion of variant DNA in a background of wild-type sequence at sensitivities approaching 5%. Perhaps one of the most astounding accomplishments of HRM analysis is the ability to detect class IV SNPs (A>T or T>A mutations) which are extremely rare (they occur at a frequency of approximately 7% withing the Human Genome) and difficult to identify due to their small melt curve temperature shift.
The attached technical note from Bio-Rad Laboratories will provide you with a fantastic overview of HRM analysis and a detailed list of things to consider prior to embarking on HRM analytical experimentation. More specifically, the technical note will cover:
- important features required for HRM compatible instrumentation
- key features for HRM compatible software
-experimental design considerations for successful HRM analysis including: HRM-compatible saturating dyes, primer design and amplicon length and PCR reaction optimization
At the tech note mentions, HRM is a low-cost, readily accessible technique that can be used to rapidly analyze multiple genetic variants. Careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. Following the attached guidelines will assist in the development of such assays.
Sean Taylor is a Field Applications Scientist at Bio-Rad Laboratories and the primary author of this tech note. Click here for other technical resources from Sean including his video entitled “A Practical Approach to MIQE for the Bench Scientist,” and the article A Simple Solution to Chromatography for High-Purity Protein Preparations: The Modular Approach