title: Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene.
abstract: BACKGROUND
  Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of CDKN2B can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable.
  
  
  RESULTS
  MS-HRM was used to assess CDKN2B methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the CDKN2B promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM.
  
  
  CONCLUSIONS
  dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.
authors.affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 8006, Australia. ida.Candiloro@petermac.org
authors.forename: Ida L M
authors.initials: IL
authors.lastname: Candiloro
authors.forename: Thomas
authors.initials: T
authors.lastname: Mikeska
authors.forename: Peter
authors.initials: P
authors.lastname: Hokland
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic