15重快速STR复合扩增体系的构建  

Construction of a 15-plex Rapid STR Multiplex Amplification System

作　　者：韩俊萍[1] 孙敬[2,3] 欧元[2,3] 刘鹏[4] 叶健[2,3] 赵雯雯[2,3] 王雪倩[2,3] 张译文[2,3] 刘耀[2,3] 李彩霞[2,3] 

机构地区：[1]中国人民公安大学刑事科学技术学院,北京100038 [2]公安部物证鉴定中心北京市现场物证检验工程技术研究中心,北京100038 [3]公安部物证鉴定中心法医遗传学公安部重点实验室,北京100038 [4]清华大学医学系统生物学研究中心,北京100084

出　　处：《法医学杂志》2016年第1期49-53,共5页Journal of Forensic Medicine

基　　金：中央级公益性科研院所基本科研业务费专项资金计划(2012JB012);公安部技术研究计划(2014JSYJA011)

摘　　要：目的建立一套15重快速STR复合扩增体系。方法选择14个常染色体基因座以及1个性别基因座,采用Fast Start Taq DNA聚合酶系统,以DNA标准品9947A为模板,通过筛选扩增条件、选择热启动酶用量、调整引物平衡、优化快速扩增程序、筛选反应缓冲液、选择反应体系以及筛选添加剂等一系列复合扩增实验,比较各条件下等位基因丢失和非特异性扩增情况。结果在以1 ng DNA为模板、0.4μL聚合酶及10×Fast Start高保真反应缓冲液构成10μL快速体系的条件下,32 min即可获得标准DNA全部15个STR基因座的完整分型,无等位基因丢失和非特异性扩增现象,等位基因均衡性良好。同时,5%甘油、0.01%明胶、0.05%明胶和5 mmol/L硫酸铵可作为PCR扩增过程中拟加入的反应添加剂。结论本研究建立的15重快速STR复合扩增体系可以明显缩短反应时间,提高样品检测效率。Objective To establish a 15-plex rapid STR multiplex amplification system. Methods Four- teen auto-chromosome loci and one sex-chromosome were selected to compare the situations of allelic losses and nonspecific amplication under different conditions. FastStart Taq DNA polymerase and DNA standard sample 9947A were used during amplification and optimization process.15-plex rapid STR am- plification system was achieved by performing various experiments including selection of amplification conditions and the volume of DNA polymerase, adjustment of inter-locus balance, optimization of rapid amplification, screening of reaction buffers, selection of reaction volume, and a variety of additives. Results Using 10 p,L rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10xFastStart high fidelity reaction buffer, a complete and well-balance DNA profile of 15 STR loci for standard genomic DNA was obtained in 32 minutes, without the allele drop-out and non-specific amplicons. Meanwhile, 5% glycerinum, 0.01% gelatin, 0.05% gelatin and 5mmol/L ammonium sulfate could be used as the re- active additive during the amplification procedure. Conclusion The 15-plex rapid STR multiplex amplifi- cation system can be used to decrease reaction time and enhance sample throughput.

关 键 词：法医遗传学 短串联重复序列 聚合酶链反应 体系建立 

分 类 号：R394.8[医药卫生—医学遗传学]