5hmC-MIQuant: Ultrasensitive Quantitative Detection of 5-Hydroxymethylcytosine in Low-Input Cell-Free DNA Samples
5hmC-MIQuant: Ultrasensitive Quantitative Detection of 5-Hydroxymethylcytosine in Low-Input Cell-Free DNA Samples
复制标题
5hmC-MIQuant：低输入无细胞 DNA 样品中 5-羟甲基胞嘧啶的超灵敏定量检测
DOI：
10.1021/acs.analchem.9b04920
复制
发表时间：
2020
影响因子：
7.4
通讯作者：
Zhang Xin-Xiang
中科院分区：
文献类型：
--
作者：
Yuan Fang;Yu Yue;Zhou Ying-Lin;Zhang Xin-Xiang
基于无细胞DNA（cfDNA）的生物标志物，如突变和甲基化，为疾病诊断和预后提供了有前途的非侵入性策略。然而，除了高通量测序之外，迄今为止还没有替代方法来检测cfDNA中的表观遗传标记，例如5-羟甲基胞嘧啶（5 hmC）。在这里，我们描述了一种基于MnO 2氧化和肼-s-三嗪试剂（i-Pr 2N）标记的方法，命名为5 hmC-MIQuant，实现了低输入DNA样品中5 hmC的超灵敏高效液相色谱-串联质谱（HPLC-MS/MS）定量。该策略使5 hmC的检测灵敏度提高了178倍，检测限低至14阿莫尔。通过简单的制备步骤，5 hmC-MIQuant可以在低至340 pg的基因组DNA（相当于57个二倍体基因组拷贝）中定量5 hmC水平。使用5 hmC-MIQuant成功分析了来自人血浆的cfDNA样品。该方法有望用于珍贵样品中5 hmC功能的鉴定和基于5 hmC的无创性疾病诊断。
Cell-free DNA (cfDNA)-based biomarkers such as mutation and methylation offer promising noninvasive strategies for disease diagnosis and prognosis. However, besides high-throughput sequencing, there has been no alternative approach to date to detect the epigenetic marks, such as 5-hydroxymethylcytosine (5hmC), in cfDNA. Here, we described a MnO2oxidation and hydrazine-s-triazine reagent (i-Pr2N) labeling based method named 5hmC-MIQuant that achieved ultrasensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) quantification of 5hmC in low-input DNA samples. This strategy improved the detection sensitivity of 5hmC by 178 times, and the limit of detection was as low as 14 amol. With simple preparation steps, 5hmC-MIQuant could quantify the 5hmC level in as little as 340 pg genomic DNA (equivalent to 57 copies of diploid genome). cfDNA samples from human plasma were successfully analyzed using 5hmC-MIQuant. This method is promising for the identification of 5hmC function in precious samples and the 5hmC-based noninvasive disease diagnosis.