应用DNA工作站进行批量血斑STR分型的研究

目的建立对大批量血斑样品PCR-STR基因分型检测的自动化新方法。方法应用自动化DNA工作站改良优化Chelex-100法和DNAIQ磁珠法的实验条件,建立两种批量血斑的自动化DNA提取方法;筛选确定PCR-STR反应体系的构建和PCR-STR产物测序电泳分析前处理程序。结果1104份血斑样品经Chelex-100法批量提取、Profiler Plus试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.43ng/ml,荧光检测信号在200~800RFU之间;对其中114份血斑样品用DNAIQ磁珠法批量提取、同试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.7ng/ml,荧光检测信号在1000~2000RFU之间;对其中50份血斑进行自动和手动Chel-ex-100检验法比较,成功率分别为100%和98%,且前者等位基因峰高信号更均衡。结论本文建立的自动化DNA工作站批量检测方法,在成功率、稳定性、均一性等方面具有优势。

PCR-STR genotyping of batches of bloodstain samples by using automatic DNA workstation

Objective To establish automatic PCR-STR genotyping method for batches of bloodstain samples.Methods Two DNA extraction methods, chelex-100 and magnetic beads methods, were improved and optimized for establishing optimal automatic DNA extraction method for batches of bloodstain samples with automatic DNA processor. In addition, new automatic procedures for PCR reaction system setup and pre-treatment of PCR products were also proposed in this article. Results 1104 bloodstain samples were successfully analyzed for all 9 STR loci by using the improved Chelex-100 DNA extraction method and Profiler plus Kit. The average concentration of DNA extracts was proved to be 0.43ng/ml, and the range of fluorescent signal of amplified products was between 200 to 800 RFU. In addition, 114 of those 1104 samples were also successfully analyzed for all 9 STR loci by using the improved magnetic beads DNA extraction method and Profiler plus Kit. The average concentration of these 114 DNA extracts was 0.7ng/ml, and the range of fluorescent signal of these amplified products was between 1000 to 2000 RFU. The comparison result between automatic and manual Chelex-100 extraction methods from 50 bloodstains samples showed that their successful rate were 100% and 98% respectively, and different alleles at the same locus can be amplified more evenly in the former.Conclusion The established DNA genotyping method for batches of bloodstain samples using automatic DNA workstation in this study was of obvious advantages at the aspect of successful rate, stability, and uniformity.