单管一步甲基化可变位点分析技术

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

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2h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。

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 dif- ferentially 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 methyla- tion-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 ~C, 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 simi- lar to that of the traditional method. The rapid classifications of the samples were also realized. Conclu- sion 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.