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In-Tube DNA Methylation Profiling by Fluorescence Melting Curve Analysis

^aAuthor for correspondence. Fax 45-35-25-77 21; e-mail perg{at}cancer.dk.

Background: Most PCR assays for detection of 5-methylcytosine in genomic DNA entail a two-step procedure, comprising initial PCR amplification and subsequent 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 amplification of bisulfite-treated DNA and melting analysis by using a thermal cycler coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR Green 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 3 °C between unmethylated and fully methylated alleles. This assay could easily distinguish patients with Prader-Willi syndrome or Angelman syndrome from individuals without these conditions. Melting curve analysis also 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^Ink4b alleles showed broadened melting peaks with overall T_ms between those of the unmethylated and fully methylated alleles.

Conclusions: Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.

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