Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B p15 geneReportar como inadecuado




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Epigenetics and Chromatin

, 1:7

First Online: 03 November 2008Received: 28 April 2008Accepted: 03 November 2008

Abstract

BackgroundMethylation-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.

ResultsMS-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.

ConclusiondMS-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.

List of abbreviations usedAMLacute myeloid leukaemia

dMS-HRMdigital methylation-sensitive high resolution melting

MS-HRMmethylation-sensitive high resolution melting

WGAwhole genome amplification.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-8935-1-7 contains supplementary material, which is available to authorized users.

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Autor: Ida LM Candiloro - Thomas Mikeska - Peter Hokland - Alexander Dobrovic

Fuente: https://link.springer.com/







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