锚定多重PCR技术的建立及其在核酸相对分子质量标准制备中的应用  

Establishment of anchored multiplex PCR and its application to preparing nucleic acid relative molecular mass standards

作　　者：严瑞芬[1,2] 屈武斌[2] 吴永红[2] 刘虎岐[1] 张成岗[1,2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]军事医学科学院放射与辐射医学研究所蛋白质组学国家重点实验室,北京100850

出　　处：《军事医学科学院院刊》2010年第6期516-518,522,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家重点基础研究发展计划(973计划)(2006CB504100);国家科技重大专项课题(2009ZX09503-002;2009ZX09301-002;2009ZX09103-616);国家自然科学基金课题(30900862;30800196;30771230)

摘　　要：目的利用5'端锚定引物及多重聚合酶链反应(PCR)技术建立高效、特异性扩增核酸片段的方法,并探讨其在核酸相对分子质量(Mr)标准(DNA标志物)制备中的应用。方法针对常用的pMD19-T载体(2692 bp)序列,利用命令行程序pd4marker.py,结合Primer3及多因素引物特异性评估软件MFEprimer,设计5'端锚定的上游引物和7对扩增不同目的片段的下游引物;其次,以转化有pMD19-T载体的大肠杆菌DH5α菌液为模板,分别进行一至七重PCR扩增;并将扩增产物经2%琼脂糖凝胶分离及凝胶成像仪采集图像分析;最后,评估该方法在核酸Mr标准制备中的应用。结果制备了特异性较好的一至七重PCR扩增产物,可针对不同实验目的进行不同核酸Mr标准的制备。结论建立了一种快速、特异性扩增核酸片段的锚定多重PCR方法,可根据不同实验目的,快速制备出不同Mr标准大小的核酸片段,进一步拓展了PCR技术的应用范围。Objective To construct an efficient and specific method for amplifying DNA fragment and to probe its application to preparing the nucleic acid relative molecular mass（Mr） standards.Methods Firstly,one 5′-end anchored forward primer and seven reverse primers of different amplicon sizes were designed for the template sequence of pMD19-T vector（2692 bp） by pd4marker.py program,which utilized Primer3 to pick candidate primers and MFEprimer to evaluate the specificity of the primers.Secondly,the E.coli DH5α that contained a pMD19-T vector was used as the template of multiplex PCR before electrophoresis of 2% agarose gel was used for analyzing the PCR products.Finally,the application of this method to preparing the nucleic acid Mr standards was evaluated.Results The different multiplex PCR products were obtained as expected and the multiplex PCR was configurable for preparing different nucleic acid Mr standards for different purposes.Conclusion A fast and specific method for the amplification of nucleic acid fragment-anchored multiplex PCR is developed and different nucleic acid Mr standards can be obtained for different purposes by this method.Furthermore,the range of application of PCR technique is expanded.

关 键 词：锚定 多重PCR 核酸扩增技术 相对分子质量标准 大肠杆菌 敏感性与特异性 

分 类 号：Q75[生物学—分子生物学]