Methylation-sensitive (MS) Hi-Res Melting® is a sensitive detection method that requires no post-PCR sample manipulation. Analysis is fast and simple and achieved by comparing melting temperature (Tm) and melting curve shape. This technique is sensitive enough to show a single methylation event within a group of multiple CpGs, and multiple CpGs can be simultaneously analyzed. In addition, high discrimination is possible because of the sensitivity of Hi-Res Melting.
We amplified a 152-bp genomic fragment of a proposed regulatory element to miRNA-195 containing five CpGs distributed throughout the amplicon. We were able to differentiate overall methylation from 10 to 100% as well as see small melting differences between 39 and 40%.
Individual CpG sites appear to affect melting curve shape as does the average methylation. We also investigated ways to overcome common challenges. First, there can be variation between replicate conversions of the same sample. Commercial kits appear to minimize this problem. Second, it is commonly believed that bisulfite-treated DNA rapidly degrades, necessitating testing within hours or days of treatment. Our results demonstrated sample stability for three months at 4°C in TE buffer. Third, small sample-to-sample temperature, volume, and PCR buffer differences can confound the interpretation of results. The use of internal melting temperature calibrators during amplicon denaturation mitigated these limitations and provided for more consistent sample interpretation.
High-resolution instrumentation and advanced dyes such as LCGreen® Plus can be used to generate a highly detailed picture of methylation within a given PCR fragment. The post-PCR technique of Hi-Res Melting can be performed on bisulfite treated genomic template. This converts 5-methylcytosine to uracil, which base pairs with adenosine during PCR and, ultimately, converts to T.
Percentage differences in GC are thus transferred into the amplicon altering Tm. Hi-Res Melting is useful for detecting sequence heterogeneity. This is because heteroduplexes created from sequence heterogeneity broaden the shape of the curves. The CpG position relative to the position within the amplicon can also affect shape. Both Tm as well as curve shape can be used in combination for epigenetic studies. Methylation is inherently heterogeneous, and Idaho Technology’s LightScanner instrumentation and software with calibration are ideally suited to discriminate fine differences in sequence heterogeneity.