法医学杂志

法医学杂志 ›› 2013, Vol. 29 ›› Issue (6): 419-424.DOI: 10.3969/j.issn.1004-5619.2013.06.005

• 论著 • 上一篇    下一篇

单管一步甲基化可变位点分析技术

岳阳阳1，2，赵贵森1，2，张  倩1，鲁  涤2，翟仙敦1，莫耀南1   

  1. （1. 河南科技大学法医学院，河南 洛阳 471003； 2. 中国政法大学 证据科学教育部重点实验室，北京 100088）
  • 发布日期:2013-12-25 出版日期:2013-12-28
  • 通讯作者: 赵贵森，男，副教授，主要从事法医遗传学研究；E-mail：lyfy@haust.edu.cn
  • 作者简介:岳阳阳（1988—），男，陕西兴平人，硕士研究生，主要从事法医分子遗传学研究
  • 基金资助:

    证据科学教育部重点实验室（中国政法大学）开放基金资助项目（08KFKT002）

One-step Methylation Variable Position Analysis Technology in Single-tube

YUE YANG-YANG1,2, ZHAO GUI-SEN1,2, ZHANG QIAN1, LU DI2, ZHAI XIAN-DUN1, MO YAO-NAN1   

  1. （1. School of Forensic Medicine, Henan University of Science and Technology, Luoyang 471003, China; 2. Key Laboratory of Evidence Science, China University of Political Science and Law, Ministry of Education, Beijing 100088, China）
  • Online:2013-12-25 Published:2013-12-28

摘要： 目的 建立单管一步甲基化可变位点（methylation variable position，MVP）分析技术——单管消化后PCR链融解曲线分析（post-digestion PCR-melting curve analysis，PDP-MCA）。 方法 以文献报道的差异甲基化区（differentially methylated region，DMR）为模型，在MVP两侧设计一组解链温度各不相同的引物。应用FastDigest甲基化敏感性限制酶（methylation-sensitive restriction enzyme，MSRE），在同一反应管内顺次进行DNA的酶切、复合扩增和MCA检测，生成MCA图谱。同时用该方法和传统的MSRE-PCR MCA技术检测相同样品（外周静脉血、精液、阴道液各5份），比较两种方法检测结果，验证其可行性，并分析比较不同样品的MCA/HRM图谱。 结果 解链温度相差2 ℃以上的片段，MCA峰分离良好，复合扩增后可以用MCA技术检测。应用单管PDP-MCA技术，可以集酶切、扩增和检测三步于一管，在2 h内得到与传统方法一致的特异性图谱和数据，并实现样品的快速分类鉴别。 结论 单管PDP-MCA技术可以实现多个MVP的单管、闭管检测，具有简便、快速、易于自动化等优点，可用于样品DNA甲基化差异的检测。

关键词: 法医遗传学, DNA甲基化, 链融解曲线, 甲基化敏感性限制酶, 甲基化可变位点

Abstract: Objective To develop the single-tube one-step methylation variable position （MVP） analysis technology——single-tube post-digestion PCR-melting curve analysis （PDP-MCA）. Methods Based on differentially methylated region （DMR） reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme （MSRE）, DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples （peripheral venous blood, semen, and vaginal fluid, 5 samples each type） were tested by single-tube one step MVP and traditional MSRE- PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. Results When the melting temperature of the fragments had a differential of 2 ℃, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions （digestion, amplification and detection） into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. Conclusion Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.

Key words: forensic genetics, DNA methylation, melting curve, methylation-sensitive restriction enzyme, methylation variable position

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