法医学杂志

法医学杂志 ›› 2019, Vol. 35 ›› Issue (2): 210-215.DOI: 10.12116/j.issn.1004-5619.2019.02.015

• 论著 • 上一篇    下一篇

REPLI-g?? Single Cell Kit应用于微量DNA扩增的效率评估

徐倩南1，2， 沈琼3， 张佳怡2，4， 张轶伦2，5， 李莉2， 刘希玲2， 李成涛1，2   

  1. 1. 温州医科大学基础医学院法医学系，浙江 温州 325035； 2. 司法鉴定科学研究院 上海市法医学重点实验室 上海市司法鉴定专业技术服务平台，上海 200063； 3. 上海交通大学医学院附属第九人民医院超声诊断科，上海 201999； 4. 内蒙古医科大学法医学教研室，内蒙古 呼和浩特 010030； 5. 内蒙古科技大学包头医学院，内蒙古 包头 014060
  • 发布日期:2019-04-25 出版日期:2019-04-28
  • 通讯作者: 李成涛，男，研究员，博士研究生导师，主要从事法医遗传学研究；E-mail：lichengtaohla@163.com
  • 作者简介:徐倩南（1994—），女，硕士研究生，主要从事法医遗传学研究；E-mail：2550038357@qq.com
  • 基金资助:
    “十三五”国家重点研发计划资助项目（2018YFC0807202）；国家自然科学基金资助项目（81871534，81625013）；中央级科研院所公益专项资助项目（GY2017G-3）；上海市标准研制资助项目（17DZ2203800）；上海市科技创新行动计划资助项目（16DZ1205500）；上海市法医学重点实验室资助项目（17DZ2273200）；上海市司法鉴定专业技术服务平台资助项目(19DZ2292700)；上海市人才项目（2017485，2017115，2018111）

Evaluating the Efficiency of REPLI-g?? Single Cell Kit for Trace DNA Amplification

XU Qian-nan1,2, SHEN Qiong3, ZHANG Jia-yi2,4, ZHANG Yi-lun2,5, LI Li2, LIU Xi-ling2, LI Cheng-tao1,2   

  1. 1. Department of Forensic Medicine, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325035, Zhejiang Province, China; 2. Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Science, Shanghai 200063, China; 3. Department of Ultrasound Diagnosis, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 201999, China; 4. Department of Forensic Medicine, Inner Mongolia Medical University, Hohhot 010030, China; 5. Baotou Medical College, Inner Mongolia University of Science & Technology, Baotou 014060, Inner Mongolia Autonomous Region, China
  • Online:2019-04-25 Published:2019-04-28

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摘要： 目的 评估REPLI-g?? Single Cell Kit应用于检材DNA扩增的效率，探讨该试剂盒在法医学微量DNA扩增方面的应用价值。 方法 选取3种微量DNA提取试剂盒，对来自10名无关个体的外周血进行DNA提取，并对所提DNA的产量和纯度进行比较。根据比较结果选取1例DNA样本进行浓缩稀释作为全基因组扩增起始样本，使用REPLI-g?? Single Cell Kit对起始样本进行全基因组扩增，计算扩增产量及扩增倍数，并进行琼脂糖凝胶电泳检测片段分布情况。使用Goldeneye?? DNA身份鉴定系统20A对起始样本和经全基因组扩增所得DNA进行STR分型。 结果 经比较后选取由QIAsymphony?? DNA Investigator?? Kit所提取的1例DNA进行浓缩稀释作为全基因组扩增起始样本。经REPLI-g?? Single Cell Kit对一系列起始样本全基因组扩增后，发现扩增产量均值最低可达到8.77×103 ng，相应的扩增倍数均值为1.40×106，DNA片段大且集中。全基因组扩增样本随着起始样本量的减少，STR分型成功率降低，但当起始样本量低于0.5 ng时，全基因组扩增后样本的STR分型成功率高于相同起始量未经全基因组扩增样本的STR分型成功率。 结论 应用REPLI-g?? Single Cell Kit可有效提高模板DNA总量，特别对于微量DNA，可在一定程度上提高STR分型成功率。

关键词: 法医遗传学, 短串联重复, 全基因组扩增, 多重置换扩增, 微量检材

Abstract: Objective To evaluate the efficiency of REPLI-g?? Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g?? Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. GoldeneyeTM DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g?? Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony?? DNA Investigator?? Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g?? Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g?? Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Key words: forensic genetics, short tandem repeat, whole genome amplification, multiple displacement amplification, trace samples