In-tube DNA methylation profiling by fluorescence melting curve analysis

Background: Most PCR assays for detection of 5-methylcytosine in genomic DN A entail a two-step procedure, comprising initial PCR amplification and sub sequent product analysis in separate operations that usually require manual transfer. These methods generally provide information about methylation of only a few CpG dinucleotides within the target sequence. Methods: An in-tube methylation assay is described that integrates amplific ation of bisulfite-treated DNA and melting analysis by using a thermal cycl er coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR G reen I) during a linear temperature transition. Results: Analysis of a region comprising 11 CpG sites at the SNRPN promoter CpG island showed that the melting temperature (T-m) differed by similar t o3 degreesC between unmethylated and fully methylated alleles. This assay c ould easily distinguish patients with Prader-Willi syndrome or Angelman syn drome from individuals without these conditions. Melting curve analysis als o allowed resolution of methylation "mosaicism" at the p15(Ink4b) promoter in bone marrow samples from patients with acute myeloid leukemia (AML). AML samples representing pools of heterogeneously methylated p15'"k4b alleles showed broadened melting peaks with overall T(m)s between those of the unme thylated and fully methylated alleles. Conclusions: Integration of PCR and fluorescence melting analysis may be us eful for simple and cost-effective detection of aberrant methylation patter ns. (C) 2001 American Association for Clinical Chemistry.