Dietrich Dimo
Institute of Pathology, University Hospital of Bonn, Bonn, Germany.
Department of Otolaryngology, Head and Neck Surgery, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105, Bonn, Germany.
Methods Mol Biol. 2018;1655:239-249. doi: 10.1007/978-1-4939-7234-0_18.
Circulating cell-free DNA (ccfDNA) can be found in various body fluids, i.e., blood (serum and plasma), urine, pleural effusions, and ascites. While ccfDNA predominantly originates from physiological processes, a fraction might be related to pathological events, e.g., cancer. Aberrant DNA methylation, which is considered a hallmark of cancer, can be assessed accurately in ccfDNA. Consequently, DNA methylation testing in body fluids represents a powerful diagnostic tool in the clinical management of malignant diseases. Frequently, however, the total amount of disease-related ccfDNA in a sample is low and masked by an excess of physiological ccfDNA. Thus, DNA methylation analysis of tumor-derived DNA is challenging, and high volumes of body fluids need to be analyzed in order to ensure a sufficient abundance of the analyte in the test sample. DNA methylation assays are usually based on prior conversion of cytosines to uracils by means of bisulfite. This reaction takes place under harsh chemical conditions leading to DNA degradation and therefore necessitates a proper DNA purification before downstream analyses. This article describes a protocol which allows for the preparation of ultra-pure bisulfite-converted DNA from up to 3 ml blood plasma and serum, which is well suited for subsequent molecular biological techniques, e.g., methylation-specific real-time PCR.
循环游离DNA（ccfDNA）可在多种体液中找到，即血液（血清和血浆）、尿液、胸腔积液和腹水。虽然ccfDNA主要来源于生理过程，但一部分可能与病理事件有关，例如癌症。异常DNA甲基化被认为是癌症的一个标志，可以在ccfDNA中准确评估。因此，体液中的DNA甲基化检测是恶性疾病临床管理中的一种强大诊断工具。然而，样本中与疾病相关的ccfDNA总量通常较低，并被过量的生理性ccfDNA掩盖。因此，肿瘤衍生DNA的DNA甲基化分析具有挑战性，需要分析大量体液以确保测试样本中有足够量的分析物。DNA甲基化检测通常基于通过亚硫酸氢盐将胞嘧啶预先转化为尿嘧啶。该反应在苛刻的化学条件下进行，导致DNA降解，因此在下游分析之前需要进行适当的DNA纯化。本文描述了一种方法，该方法允许从多达3毫升血浆和血清中制备超纯亚硫酸氢盐转化的DNA，这非常适合后续的分子生物学技术，例如甲基化特异性实时PCR。
