6个Y-STR荧光复合扩增系统的建立及应用

Development of six Y-STR fluorescent-multiplex amplification system and its application

目的建立6个Y-STR荧光复合扩增体系,评价其法医学应用价值。方法设计DYS444,GATA-A7.2,GATA-A10,DYS390,GATA-A7.1,DYS443荧光复合扩增引物,扩增总体积20μL,内含模板DNA0.5～10ng,PCR产物用3130遗传分析仪电泳,GenemapperID v 3.2分析结果,根据等位基因标准命名各等位基因,并评价该系统的特异性、准确性、均衡性、灵敏度及对混合血样的分析能力。结果当dNTP、Mg2+分别为200μmol/L、1.5mmol/L时扩增效果最佳,0.5～10ngDNA模板量均能获得较好的扩增效果,各基因座分型结果清晰,扩增均衡,特异性强,重现性好,基本满足实际应用的性能要求。对湖北汉族群体进行遗传学调查,结果GD值0.580 3～0.722 3,共检出158种单倍型,其多样性为0.995 8。结论本文6个Y-STR扩增系统分型可靠,配合常用Y-STR分型试剂盒,可进一步提高个体识别能力。

Objective To develop a fluorescent-multiplex amplification system for six Y-STR loci and evaluate its application value in forensic medicine.Methods Multiplex amplification primers of six Y-STR loci（DYS444,GATA-A7.2,GATA-A10,DYS390,GATA-A7.1 and DYS443） were designed.The total volume of amplification system was 20μL,including 0.5～10ng of DNA template.Electrophoresis of PCR production was carried out on 3130 genetic analyzer.The result was analyzed with Genemapper ID v 3.2 software.Every allele was named according to the allelic ladder.The properties of the amplification system were evaluated,including its specificity,accuracy,equilibrium,sensitivity of amplification and analysis ability for mixed samples.Results The best amplification result could be attained from 0.5～10ng DNA template or in the condition of 200μmol/L of dNTP and 1.5mmol/L of Mg^2＋ in amplification system.The alleles of six Y-STR loci were successfully tested,showing unambiguous genotyping,amplification equilibrium,strong specificity and good reproducibility.The amplification system could satisfy the need of forensic practice.In study of population genetics for Han population in Hubei province,the gene diversity values（GD） for each locus ranged from 0.580 3 to 0.722 3.158 kinds of haplotypes were identified and the haplotype diversity（HD） was 0.995 8.Conclusion The six Y-STR fluorescence multiplex amplification system is reliable,and can further improve the discrimination power combined with other commercial Y-STR typing kits.