单管直扩38重祖先信息InDels体系的构建及其在微流控芯片系统的应用  

A Single-tube Direct Amplification 38-plex Ancestry Informative InDels Assay and Its Integration With a Microfluidic System

作　　者：韩俊萍[1,2] 赵蕾 王庆国[3] 江丽 谢何鑫[1] 庄斌 赵丽健 李唐松 吴瑾 李彩霞[1] HAN Jun-Ping;ZHAO Lei;WANG Qing-Guo;JIANG Li;XIE He-Xin;ZHUANG Bin;ZHAO Li-Jian;LI Tang-Song;WU Jin;LI Cai-Xia(Key laboratory of Forensic Genetics,Beijing Engineering Research Center of Crime Scene Eridence Examination,National Engineering Laboratory for Forensic Science,Instiute of Forensic Science,Beijing 100038,China;Technology Department of Chaoyang Sub-bureau,Bejing Public Securiy Bureau,Beijing,100025,China;Shandong First Medical University,Jinan 250000,China;Bejjing CapitalBio Technology Ud.Co,Bejing 101111,China)

机构地区：[1]公安部物证鉴定中心法医遗传学公安部重点实验室北京市现场物证检验工程技术研究中心现场物证溯源技术国家工程实验室,北京100038 [2]北京市公安局朝阳分局,北京100025 [3]山东第一医科大学,济南250000 [4]北京博奥晶典生物技术有限公司,北京101111

出　　处：《生物化学与生物物理进展》2020年第9期968-981,共14页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81772027);中央级公益性科研院所基本科研业务费专项资金计划(2018JB037)资助项目。

摘　　要：本文基于插入-缺失多态性(insertion-deletion polymorphism,InDel)遗传标记,从相关文献和dbSNP库中筛选出38个在东亚、欧洲、非洲人群中具有等位基因频率差异的祖先来源InDel位点,同时整合Amelogenin位点和Y染色体STR(DYS439)位点,以辅助未知样本的性别鉴定,采用PCR-CE技术构建了可以区分三大洲际人群的单管直接扩增复合检测体系,该体系可与微流控芯片系统相结合,1.7 h内完成DNA样本自动化扩增和电泳分型,利用公共数据库1000Genomes中16个人群1 607名个体的分型数据对筛选的38个InDel位点进行初步评估;同时对构建的38-plex InDels体系的灵敏度、准确性、直接扩增能力进行验证评估;检测5种不同人群的779份样本和215份唾液卡、血卡的直接扩增样本,采用聚类分析和主成分分析方法,评价体系的推断准确性.结果表明该38-plex InDels复合检测体系分型准确,灵敏度达157 pg,能够对唾液卡和血卡直接扩增,可以区分三大洲际人群及其混合人群,能够准确推断待测样本的族群来源、估算祖先成分,且通过集成细胞裂解步骤将有望2h实现"样本进-结果出"式快速自动化InDel分型.In this paper,38 ancestry informative insertion-deletion(InDel) polymorphism markers,with allele frequency differences between three major continental population groups(East Asian,European,and African)were selected from the related literatures and dbSNP database.The Amelogenin locus and a Y chromosomal STR(DYS439) locus were integrated to aid in the sex determination of an unknown sample.All markers were designed and amplified in a single-tube and performed direct amplification assay based on the PCR-CE technology for inferring ancestry of three continental populations.This assay was integrated with a microfluidic system for automatic amplification and InDel genotyping using DNA samples in 1.7 h.The genotypes of 1 607 individuals obtained from the 1000 Genomes Phase Ⅲ panel were used to assess the differentiation capacity of this 38-plex InDels assay.Then,this assay was validated by sensitivity,genotyping accuracy,and direct PCR ability.779 samples from 5 populations and 215 direct amplification from saliva and blood samples were genotyped by this assay,then using the structure cluster analysis and principal component analysis to infer ancestral origins and evaluate the accuracy of the system.The results indicate that the 38-plex InDels assay can provide an accurate genotyping,with a sensitivity of 157 pg,and enable the direct amplification of DNA from saliva and blood on filter paper without sample purification.In addition,this assay is sufficient to distinguish between three continental populations and the admixture of Eurasian populations,and also accurately infer the ancestry origins of testing DNA samples.The integration of cell lysis into the microfluidic system is possible to yield ’sample-inprofile-out’ results of InDel genotyping in 2 h.

关 键 词：法医物证学 插入-缺失多态性 祖先信息位点 微流控芯片 

分 类 号：R89[医药卫生—法医学] D919.2[医药卫生—临床医学]