法医学杂志

法医学杂志 ›› 2016, Vol. 32 ›› Issue (1): 49-53.DOI: 10.3969/j.issn.1004-5619.2016.01.011

• 技术与应用 • 上一篇    下一篇

15重快速STR复合扩增体系的构建

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   

  1. （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）
  • 发布日期:2016-02-25 出版日期:2016-02-28
  • 通讯作者: 刘耀，男，研究员，主要从事法医毒物分析研究；E-mail：liuyaol123@yahoo.cn 李彩霞，女，副主任法医师，主要从事法医物证学研究及鉴定；E-mail：licaixia@tsinghua.org.cn
  • 作者简介:韩俊萍（1986—），女，博士研究生，主要从事法医遗传学研究；E-mail：pgww19861025@163.com
  • 基金资助:

    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）

Construction of a 15-plex Rapid STR Multiplex Amplification System

HAN JUN-PING1, SUN JING2,3, OU YUAN2,3, LIU PENG4, YE JIAN2,3, ZHAO WEN-WEN2,3, WANG XUE-QIAN2,3, ZHANG YI-WEN2,3, LIU YAO2,3, LI CAI-XIA2,3   

  1. （1. School of Criminal Science and Technology, Chinese People’s Public Security University, Beijing 100038, China; 2. Beijing Engineering Research Center of Crime Scene Evidence Examination, Institute of Forensic Science, Ministry of Public Security, Beijing 100038, China; 3. Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing 100038, China; 4. Medical System Biology Research Center, School of Medicine, Tsinghua University, Beijing 100084, China）
  • Online:2016-02-25 Published:2016-02-28

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FastStart 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、0.4 ?滋L聚合酶及10×FastStart高保真反应缓冲液构成10 ?滋L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。

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

Abstract: Objective To establish a 15-plex rapid STR multiplex amplification system. Methods Fourteen 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 amplification 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 μL rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10×FastStart 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 5 mmol/L ammonium sulfate could be used as the reactive additive during the amplification procedure. Conclusion The 15-plex rapid STR multiplex amplification system can be used to decrease reaction time and enhance sample throughput.

Key words: forensic genetics, short tandem repeat, polymerase chain reaction, system establishment

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