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A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

机译：HM-PCR / SNuPE组合方法可高灵敏度检测稀有DNA甲基化

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Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.
机译：背景DNA甲基化变化被广泛用作癌症检测中的早期分子标记。从循环体液或复杂组织样本中提取的DNA中罕见的甲基化变化的灵敏检测和分类对于理解肿瘤病因，临床诊断和治疗至关重要。在本文中，我们描述了一种结合的方法来监测甲基化肿瘤DNA在非肿瘤细胞的过量未甲基化背景DNA中的存在。该方法结合了重甲基-PCR，该PCR有利于从亚硫酸氢盐处理的DNA优先扩增甲基化的标记序列，并通过离子对，反相高效液相色谱分离监测甲基化特异性的单核苷酸引物延伸。结果该组合方法可检测到14 pg（即4至5个基因组拷贝）的甲基化染色体DNA，其含量为未甲基化染色体背景的2000倍（即50 ng），分析灵敏度> 90％。我们概述了已知癌症标记物（SEPT9和TMEFF2）的两个例子，用于联合测定的详细协议，并讨论了测定设计和数据解释的一般方面。最后，我们提供了一个应用实例，用于快速检测一小群结直肠癌患者血浆中的血浆DNA中的肿瘤甲基化。结论该方法可以非常准确地检测罕见的DNA甲基化，例如在体液或来自细胞或组织的DNA分离物中。该应用程序结合了标准技术，可以轻松地适应任何感兴趣的目标区域。它不需要昂贵的试剂，可用于常规筛查许多样品。

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