Methylation Analysis Using Methylation-Sensitive HRM and DNA Sequencing.
DNA methylation is a key epigenetic mechanism regulating gene expression and chromatin structure.
MS-HRM followed by Sanger-based DNA sequencing is a fast, simple method for methylation analysis that can measure methylation levels as low as 0.1 to 2%.
MS-HRM analysis offers a simple method for quickly analyzing the methylation status of specific genetic loci. Reaction products that merit further analysis can then be sequenced directly to identify precise methylation patterns.
Applied Biosystems provides an integrated set of tools for locus-specific DNA methylation analysis using an optimized, streamlined procedure.
Figure 1 (Right): MS-HRM Standard Curves With Uniform Resolution From 0% to 100% Methylation.
Methylation Analysis Experimental Workflow
The first step of the workflow is to treat genomic DNA with bisulfite, which deaminates unmethylated cytosines (C) to form uracil (U), but does not react with methylated cytosine bases. Thus bisulfite conversion changes the DNA sequence based on the methylation status of individual nucleotides in genomic DNA, and these changes can be detected via HRM analysis.
Cells-to-CpG™ Bisulfite Conversion Kit
Quick Reference Card
HRM Methylation Study
MeltDoctor™ High Resolution Melt Reagents