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首页> 外文期刊>Epigenetics & Chromatin >Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene
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Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene

机译：使用数字甲基化敏感的高分辨率熔解技术快速分析异质甲基化的DNA：应用于CDKN2B（p15）基因

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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. Conclusion 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.
机译：背景甲基化敏感的高分辨率熔解（MS-HRM）方法能够通过其特有的熔解曲线识别异质甲基化序列。为了进一步分析异质甲基化的序列，我们对MS-HRM采用了一种数字方法（dMS-HRM），该方法涉及在有限稀释后定量扩增单个模板并确定甲基化程度。我们使用这种方法来研究CDKN2B（p15）细胞周期进程抑制剂基因的甲基化，该基因在骨髓谱系的血液系统恶性肿瘤中被DNA甲基化所灭活。其启动子区通常显示异质甲基化，很少被完全甲基化。 CDKN2B的甲基化状态可用作对治疗反应的生物标记。因此，需要对其甲基化的准确表征。结果MS-HRM用于评估急性髓细胞白血病（AML）样本中的CDKN2B甲基化。在CDKN2B启动子（40/93）处甲基化的所有AML样品均表现出不同程度的异质甲基化。选择了六个代表性样品进行进一步研究。 dMS-HRM用于同时计数甲基化等位基因并评估甲基化程度。所选dMS-HRM产品的直接测序用于确定确切的DNA甲基化模式，并确认dMS-HRM估计的甲基化程度。结论dMS-HRM是分析CDKN2B和其他异质甲基化基因中甲基化的有力技术。它消除了针对甲基化或非甲基化DNA的PCR和克隆偏倚。潜在的复杂信息被简化为数字输出，从而可以对甲基化和未甲基化的等位基因进行计数，并提供给定基因座处甲基化的总体情况。下游测序被最小化，因为dMS-HRM充当筛选，仅选择甲基化克隆进行进一步分析。

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