6FAM-HEX-TMR-ROX四色荧光分析体系的构建

Construction of the matrix for fluorescence STR analysis with 6FAM-HEX-TMR-ROX

目的 为在310基因分析仪上进行6FAM—HEX—TMR—ROX四色荧光STR复合扩增分型建立基础条件。方法 以pGEM载体作为靶DNA，设计引物扩增获得500～80bp的13个长度不等DNA片段，以这些片段纯化物作为模板DNA，分别扩增获得6FAM、HEX、TMR和ROX标记的Matrix标准物，用4种Matrix标准物在310基因分析仪上构建可用于6FAM-HEX-TMR-ROX四色荧光分析的Matrix文件。结果 扩增所得的13个非标记片段，长度分别为80、100、120、140、160、180、200、220、260、300、340、400、500bp，在非变性聚丙烯酰胺凝胶连续电泳-银染凝胶上均表现为单条带，6FAM、HEX、TMR、ROX分别标记的片段通过310基因分析仪检测均为单峰。混合ROX标记的80、120、180、220、260、300bp的6个片段获得的内标，显示出良好的线性关系。结论 建立了有效的6FAM-HEX-TMR-ROX四色荧光分析Matrix，为进行多个STR基因座荧光标记复合扩增检测奠定了基础。

Objective To construct the matrix of four fluorescence color analysis system for STR genotyping based on ABIPRISM 310 Genetic Analyzer. Methods The PCR primers using pGEM vector as target DNA were designed to obtaining thirteen DNA fragments, which lengths were between 80 -500bp. FAM, HEX, TMR and ROX were employed to label primers, respectively. DNA fragments labeled with fluorescence were obtained by PCR with the primers. Matrix sample was prepared by mixing the thirteen DNA fragments labeled with same fluorescence. New matrix file was constructed through electrophoresis of FAM, HEX, TMR and ROX labeled matrix sample on ABIPRISM 310 Genetic Analyzer. Results Thirteen DNA fragments with 80.100.120.140.160.180,200.220.260.300.340bp,400bp and 500bp were obtained with PCR techniques. Each of the non - fluorescence labeled fragment showed a single band in the PAG - electrophoresis, while the fluorescence labeled DNA fragment displayed a single peak in ABIPRISM 310 Genetic Analyzer. A ROX labeled internal lane standard with six DNA fragment, including 80. 120. 180. 220. 260 and 300bp, was obtained. Conclusion A new matrix file was constructed. It was useful for determining more than 3 STR loci using ABIPRISM 310 Genetic Analyzer. The ROX labeled internal lane standard with good linearity provided an approach satisfied with STR typing in Forensic laboratory.