High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection typing using inexpensive generic heteroduplex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene (RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two different heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identified by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP, and genotyping based on high-resolution amplicon melting.

The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

DNA methyltransferase 3A (DNMT3A ) is mutated in a subset of de novo acute myeloid leukemia patients and is associated with poor overall and event-free survival. Because routine Sanger sequencing of the 23 DNMT3A exons is impractical in clinical laboratories, we developed a high-throughput method using high-resolution melting (HRM) analysis, which identifies sequence variants by detecting subtle changes in the melting patterns of mutant DNA in comparison with WT sequences. DNA from 104 acute myeloid leukemia patients was tested for mutations in 12 exons encoding 3 major functional domains of DNMT3A : the PWWP (proline-tryptophan-tryptophan-proline) domain (exons 8 to 10), the ADD (ATM-DNMT3-DNMT3L) zinc finger, and the methyltransferase domains encoded by exons 15 to 23. HRM analysis identified 20 of 104 patient samples as variants, which we confirmed by Sanger sequencing. Codon 882 of exon 23 was mutated at the highest frequency with an occurrence rate of 11.5%. All HRM WT calls were confirmed to be devoid of mutations by Sanger sequencing. We also identified seven novel and previously unreported DNMT3A mutations. Structural modeling showed seven of the eight missense mutations detected in our study increased the free energy, destabilized protein, and altered solvent accessibility, suggesting their loss-of-function nature. These data demonstrate HRM analysis to be a higher throughput, sensitive, and efficient alternative to Sanger sequencing for detecting DNMT3A mutations in the clinical diagnostic laboratory.

We describe a rapid, high-throughput method to scan for new RNA editing sites. This method is adapted from high-resolution melting (HRM) analysis of amplicons, a technique used in clinical research to detect mutations in genomes. The assay was validated by the discovery of six new editing sites in different chloroplast transcripts of Arabidopsis thaliana . A screen of a collection of mutants uncovered a mutant defective for editing of one of the newly discovered sites. We successfully adapted the technique to quantify editing of partially edited sites in different individuals or different tissues. This new method will be easily applicable to RNA from any organism and should greatly accelerate the study of the role of RNA editing in physiological processes as diverse as plant development or human health.