Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing
Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing
复制标题
DOI：
10.1021/ac061961k
复制
发表时间：
2007-03-01
影响因子：
7.4
通讯作者：
Mathies, Richard A.
中科院分区：
文献类型：
--
作者：
Liu, Peng;Seo, Tae Seok;Mathies, Richard A.
研制了一种便携式法医遗传分析系统，该系统由用于短串联重复序列(STR)片段扩增和分离的微流控装置以及用于芯片操作和四色荧光检测的仪器组成。该微型设备在160毫升的小室中进行聚合酶链式反应(PCR)，并在7厘米长的分离通道中进行毛细管电泳(CE)。该仪器设计集成了聚合酶链式反应热循环、电泳分离、气动阀门流体控制和四色激光激发荧光检测。建立了由釉原蛋白和三个Y-STR基因座(DYS390、DYS393、DYS439)组成的四重Y-STR分型系统，并用于验证研究。在1.5h内完成了对这4个基因座的多重扩增、35个循环的CE分离和4色荧光检测，所有的扩增产物都可以被检测到，最低检测限为20个拷贝的雄性标准DNA。还成功地对口腔拭子和从案件证据中提取的人骨进行了现实世界的法医分析。混合分析表明，即使在男女模板比例为1：10的情况下，也可以获得平衡的DNA图谱。该便携式PCR-CE系统的成功开发和运行为法医案件、大规模灾难样本或安检点个人的快速DNA分型奠定了可行性。
A portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.