High-resolution melting for accurate assessment of DNA methylation.

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 cytosine as thymine (4 ). High-resolution mapping of individual CpG sites by bisulfite genomic sequencing may still be technically challenging and labor-intensive, however, and may not always be the most rational approach to methylation analysis. A large number of simpler PCR-based assays have been developed that use bisulfite-converted DNA as a template and provide information on DNA methylation with varying sensitivity, specificity, and extent of detection (5, 6). One of these assays, methylation-specific melting curve analysis, was first described by Worm et al. (7 ) in 2001 and has now been further developed on a highresolution melting platform by White et al. (3 ). The principle of this method is that PCR products generated from bisulfite-treated DNA templates with different contents of methylcytosine show differences in melting temperature (Tm), which can be resolved by melting analysis in a thermal cycler coupled with a fluorometer (7–9). DNA melting is the cooperative unwinding of the double-helical structure into single-stranded random coils. The Tm of a DNA molecule can be determined by gradual heating of the DNA in aqueous solution, and it is highly dependent on the nucleotide sequence; a singlebase substitution may change the Tm of a PCR product by up to 1 °C, depending on the length of the amplified sequence and the type of substitution. In melting experiments dating back 25 years, single-base mutations were detected in a spectrophotometer by monitoring ultraviolet absorbance of DNA solutions near or at 260 nm while slowly increasing the temperature (10 ). Now, a new generation of PCR cyclers has paved the way for successful high-throughput, in-tube melting analysis. Inclusion in the reaction of a PCR-compatible fluorescent dye that specifically binds to double-stranded DNA allows the melting properties of a PCR product to be examined immediately after amplification (11 ). During a linear temperature transition, an abrupt decrease in fluorescence reflects the cooperative melting of the PCR product. This feature was introduced as a simple means to distinguish between specific and nonspecific PCR products, but detection of single-base substitutions has now become possible with the use of instruments specifically designed for high-resolution melting in combination with special saturation dyes (12 ). Resolution of DNA methylation by melting analysis relies on the fact that the Tm of a PCR product generated from bisulfite-treated DNA reflects the methylation status of the original DNA template (8 ). Because unmethylated cytosines will be converted into uracil during bisulfite treatment and subsequently amplified as thymine, whereas methylcytosines will remain as methylcytosine and be amplified as cytosine, the methylated sequence will have a higher G:C content, and hence a higher Tm, than the corresponding unmethylated sequence. After amplification with primers that will not differentiate between methylated and unmethylated molecules, the melting properties of the PCR products can be examined in the thermal cycler by slowly elevating the temperature under continuous or step-wise fluorescence acquisition. The melting curves or derived melting peaks provide a profile of the methylation status of the entire pool of DNA molecules in the sample (7, 8). White et al. (3 ) have established a simple diagnostic assay for Prader-Willi syndrome and Angelman syndrome. Both syndromes are neurodevelopmental disorders caused by genomic abnormalities at an imprinted region on chromosome 15q11.2, leading to methylation changes at the small nuclear ribonucleoprotein polypeptide N (SNRPN) locus (13 ). After bisulfite conversion of DNA, an approximately 240-bp sequence of the SNRPN promoter containing 21 CpG sites was amplified and subjected to high-resolution melting. Melting curves and difference plots clearly showed a biphasic melting profile for samples from healthy individuals, representing the unmethylated and methylated SNRPN alleles. In contrast, samples from individuals with Angelman syndrome showed a monophasic low-melting profile consistent with an unmethylated SNRPN promoter, and samples from individuals with Prader-Willi syndrome showed a monophasic high-melting profile consistent with a hypermethylated SNRPN promoter. Use of the automated Editorial