High resolution melt analysis is an excellent way to identify samples that fall into the same group. Examples where HRM analysis is applicable are identification of two or more members of closely related sequences (species of a common genus or closely related alleles of the same transcript), methylation, SNPs and more. If necessary, one member of each group can be sequenced to make specific sequence identification. HRM analysis is based on a saturating dye and the altered melt signature between 2 or more closely related sequences differing by as little as a single base. PCR amplicons are best if short, up to 250 bases but can be longer depending on the sequences being interrogated. The core director can help with questions. HRM is performed on our Roche LC480 real-time qPCR instrument and analyzed with provided software.
Samples- DNA samples should be provided at 25 ng/µl in nuclease-free H2O. It is critical that the samples be as clean of RNA or protein contaminants as possible as any contaminant could affect the melt characteristics of the sample DNA. Samples should be provided in the same tubes as described for RNA and DNA quantification.
Data analysis- Data will be provided in an Excel workbook. All melt curve figures and sample identifications will be provided.