Comparison of three different techniques of human sperm DNA isolation for methylation assay
Comparison of three different techniques of human sperm DNA isolation for methylation assay
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DOI：
10.1007/s11596-015-1532-0
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Hong-fang Yuan;M. Kuété;Li Su;Fan Yang;Zhiyong Hu;Bo-Zhen Tian;Hui-ping Zhang;K. Zhao
中科院分区：
文献类型：
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作者：
Hong-fang Yuan;M. Kuété;Li Su;Fan Yang;Zhiyong Hu;Bo-Zhen Tian;Hui-ping Zhang;K. Zhao
人精子DNA是重要的遗传和表观遗传物质，其染色质结构与体细胞不同。因此，用于体细胞DNA提取的常规方法可能不适合于获得精子DNA。本研究对改良硫氰酸胍法（A法）、传统酚-氯仿法（B法）和田根试剂盒法（C法）三种精子DNA提取方法进行了比较和评价。分光光度法和琼脂糖凝胶电泳分析表明，A法提取的DNA量和纯度均高于B法和C法（P<0.01）。PCR结果显示，方法A在扩增DEAD-box多肽4（DDX 4）和拷贝数变异（CNVs）方面比方法B和C更可靠，后者产生假阳性错误。精子DNA甲基化检测结果进一步表明，方法A和B均有效，且前者的定量准确度较高。结论：改良硫氰酸胍法可获得高质量、可靠的精子DNA，是一种较理想的精子DNA甲基化检测方法。
Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.