基于改进的重叠延伸聚合酶链式反应技术快速制备DNA分子质量标准  

Straightforward method for preparing a user-customizable DNA marker by improved overlap extension polymerase chain reaction

作　　者：张圆圆 颜焰 金玉兰[1] 董伟仁 周继勇[1] ZHANG Yuanyuan;YAN Yan;JIN Yulan;DONG Weiren;ZHOU Jiyong(College of Animal Sciences,Zhejiang University, Hangzhou 310058, China)

机构地区：[1]浙江大学动物科学学院

出　　处：《浙江大学学报（农业与生命科学版）》2019年第3期272-277,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点研发计划(2016YFD0500102);浙江大学实验技术研究项目(SJS201811)

摘　　要：采用改进的重叠延伸聚合酶链式反应(polymerase chain reaction,PCR)技术,简便、快速地制备DNA分子质量标准。首先,利用普通PCR技术扩增5 和3 两端序列一致的DNA片段。随后,在重叠延伸PCR的变性和退火过程中,2个单链的3 端互补序列可形成双链,且这些单链片段可同时作为模板和引物,在PCR延伸过程中延长片段。随着反应循环数的增加,小片段产物逐渐减少,大片段产物逐渐增多。取不同循环数的重叠延伸PCR产物进行琼脂糖凝胶电泳检测,根据电泳结果选择最佳的PCR循环数或不同循环数的最佳组合,无须回收纯化,即可得到分子质量相差100bp的DNA分子质量标准。经琼脂糖凝胶电泳检测,所制备的DNA分子质量标准条带清晰、分子质量准确、条带均一性好,可用于标记DNA分子质量大小。总之,本研究用于DNA分子质量标准制备的方法操作简单,周期短,成本低,无副产物,且能够根据具体需求定制DNA分子质量标准的大小。DNA marker is a set of standards that are used to indicate the approximate molecular-mass size of certain DNA fragment which separated by agarose gel electrophoresis. In the present study, a straightforward method for preparing a user-customizable DNAmarker by an improved overlap extension polymerase chain reaction (PCR) was established. The DNA fragment, containing the identical sequence in its 5′ and 3′ ends, was amplified by a conventional PCR. In the following denaturation and annealing process performed by the overlap extension PCR, two single-stranded DNA in the 3′ end of which had complementary base sequences could form double-stranded DNA. In the extension process, these single-stranded DNA was utilized as both template and primer. The number of small fragments of DNA decreased and the number of large fragments of DNA increased gradually with the increased number of PCR cycles. The products of different cycles of overlap extension PCR were taken for agarose gel electrophoresis. The optimal PCR cycle or optimal combination of different PCR cycles was chosen according to the results of electrophoresis. Then 100 bp DNA ladder marker was obtained by the overlap extension PCR under the chosen condition. There was no need for purification of PCR products as the amplified fragments could be directly used in the agarose gel electrophoresis. The bands of prepared DNA marker were clear and accurate, and could be used for molecular studies. In sum, this method for DNA marker production is simple, time saving, cost effective, byproduct free and user-customizable in comparison with current ones widely used in most laboratories.

关 键 词：DNA分子质量标准 重叠延伸聚合酶链式反应 DNA电泳 

分 类 号：Q78[生物学—分子生物学]