High-Resolution Melting for Accurate Assessment of DNA Methylation

Abstract

Methylation of cytosine in DNA is an epigenetic mark that is important for genome stability, transcriptional regulation of endogenous genes, and permanent silencing of transposable elements and viral sequences. Methylcytosine, sometimes referred to as the 5th base of DNA, constitutes 3%–6% of all cytosines in the human genome and occurs almost exclusively within the context of CpG dinucleotides (1). DNA methylation patterns have been shown to correlate with various disease states, including inherited disorders and cancer, and the identification of aberrantly methylated genes is a promising strategy in research, diagnostics, and therapeutics (2). In the current issue of Clinical Chemistry , White et al. (3) report a simple closed-tube PCR assay that combines amplification of bisulfite-treated DNA with high-resolution amplicon melting for sensitive and high-throughput assessment of DNA methylation. One major problem inherent in gene-specific DNA methylation analysis is that methylation marks are erased during conventional PCR and cloning procedures because methylcytosine is replaced with cytosine. To circumvent this problem, DNA can be treated with sodium bisulfite, which converts unmethylated cytosines to uracil, whereas methylcytosine is protected against this modification (4). The bisulfite-modified DNA can be used as template in a standard PCR to amplify specific DNA sequences and examine their methylation content. The most accurate methylation profiling can be achieved by sequence analysis of the PCR products, which display methylcytosine as cytosine and unmethylated …