19个常染色体与4个Y染色体STR复合扩增体系的建立

目的建立19个常染色体STR及Amelogenin和4个Y染色体STR基因座复合扩增体系,并对其效能进行评估。方法用五色荧光标记20＋4Y—STR基因座,建立同步扩增检测体系,用ABI3130XL遗传分析仪对扩增产物进行电泳,GeneMapperID3．2软件进行基因分型;检测体系的灵敏度、均衡性、稳定性、特异性、同一性和稳定性,并观察混合、降解及微量检材的分型情况。结果采用本文体系,DNA模板量在0．05～1．00ng时,分型准确,均衡性、特异性好;混合、降解及微量检材分型正确。该19个常染色体STR基因座的累计个人识别率大于0．999999999,三联体累计非父排除率达0．999999985,Y—STR单倍型多态性为0．592。结论本文建立的复合扩增体系分型准确,稳定,在法医学案件检验及数据库建设等方面有良好的应用前景。     

印记基因H19上游高甲基化区SNPs多态性研究

目的建立简单、高效的DNA甲基化标记和SNPs联合检测技术，并用于H19基因上游高甲基化区两组SNPs群体遗传学检测。方法用PCR—DGGE技术对232例武汉汉族无关个体H19基因上游启动子区H19FR1和H19FR2单倍型进行检测；同时选用两种甲基化敏感的限制酶（msRE）HpaⅡ和HhaⅠ，检测H19FR等位基因亲代来源。结果H19FR1区检出5种单倍型、9种表型组合，其个体识别能力（DP）、多态性信息含量（PIC）和非父排除率（PE）分别为0．803、0．58和0．322；H19FR2区检出2种单倍型、3种表型组合，其DP、PIC和PE值分别为0．626、0．37和0．162。测序结果显示，片段H19FR1含有a7342g、a7357g和g7547a3个SNPs与1个g7351c点突变；H19FR2仅含aS097g1个SNP。msREHpaⅡ或HhaⅠ可消化个体母源等位基因，PDP-DGGE分析仅能检测到父源等位基因。结论PDP-DGGE是一种简单、灵敏、高效的DNA甲基化标记和SNPs联合分析技术，其在进行多态性分型同时还可以确定等位基因的亲代来源，具有较高的法医学应用价值。     

6个五核苷酸STR基因座荧光复合扩增体系的建立

目的 构建6个五核苷酸STR基因座荧光复合扩增体系。方法筛选6个多态性程度较高的五核苷酸STR基因座D10S2325、Penta B、Penta W、PentaX、Penta D和PentaE,按照复合扩增引物设计要求,重新设计引物并标记荧光染料,经反复调整和优化,构建6基因座荧光复合扩增体系,并用该复合扩增体系对239名武汉汉族无关个体进行分型。结果6个五核苷酸STR基因座荧光复合扩增体系分型稳定,可重复性好,与各自相应单基因座分型结果完全一致;累积个人识别率达0．999999988,累积非父排除率达0．998063807。结论本文构建的6个五核苷酸STR基因座荧光复合扩增体系具有很高的法医学实用价值,可作为商品化试剂盒的有效补充。     

27-plex SNPs复合扩增检测体系构建与应用评价

目的建立27个常染色体SNPs复合检测体系,用于未知个体种族来源推断。方法通过对Hap Map数据库中描述祖先(非洲、欧洲、东亚)的遗传标记信息分析,选出27个SNPs位点,构建27个SNPs复合扩增体系;采用该体系对17个不同祖先人群的1 164份样本进行测试,得到的分型数据和在Hap Map数据库查询到的11个相关人群的数据;采用据聚类分析方法(K=3)进行祖先成分和匹配率计算,分析推算样品9947A的祖先来源,并进行体系性能验证。结果该体系可以进行单一和混合人群的种族来源和种族成分推断,来自新疆的人群遗传成分呈现在欧洲与东亚祖先之间连续分布,样品9947A祖先成分和匹配率与相关文献分析结果一致。浓度最低为0.1ng/μL时27个等位基因均可正确判型。结论本文构建的27-plex SNPs复合体系可以精确推断非洲、欧洲、东亚血统的个体祖先起源,且对欧亚混合人群(欧洲/东亚)有较好的推断能力,可在相关研究和实践中选择使用。     

16个线粒体DNASNP复合检测体系的建立

目的建立一种复合检测线粒体DNA（mtDNA）单核苷酸多态性（SNP）分型的方法。方法通过设计等位基因特异性引物并结合毛细管电泳分型技术平台,建立包含16个mtDNASNP位点的复合扩增检测体系。对50名汉族无关个体血样进行mtDNASNP检测,并通过直接测序法对其分型结果进行验证。结果50个样本经本检测体系复合扩增后,均得到清晰的SNP分型结果,当模板量在0．5～10pg时能得到较理想的分型图谱。样本的复合检测结果与直接测序法结果完全一致。结论建立的复合检测体系检测mtDNASNP方法灵敏度高、操作简便、分型准确,为针对mtDNA进行简便、有效的中高通量多态性分型提供了一种新方法。     

个体识别SNPs位点组合筛选与法医学应用价值初探

目的筛选用于包括中国主要民族在内的多个群体个体识别的SNPs位点组合体系。方法以Kidd实验室筛选的86个SNPs位点、欧洲SNPforID组织构建的52-plex SNPs复合检测体系为基础,收集和整理这些位点在HapMap数据库中11个人群的分型数据,计算各位点杂合度和Fst值,筛选杂合度〉0.4,Fst值〈0.06,并在研究人群中处于Hardy-Weinberg和连锁平衡的位点组合。针对这些位点,采用MassARRAY分子阵列技术对自行收集的8个人群（尼日利亚人、坦桑尼亚查加人、印度人、丹麦人、俄罗斯汉特人、中国汉族、藏族、维吾尔族）308份样本进行分型,统计群体遗传学参数。结果按本文标准共筛选出66个SNPs位点,均符合Hardy-Weinberg平衡,之间互不连锁,平均杂合度和Fst值分别为0.475、0.014。在本文收集的8个人群中的随机匹配概率在1.45E-24~4.72E-27之间,累积非父排除率为0.999 995 608~0.999 997 876之间。结论本文筛选的SNPs组合系统具有较强的个体识别能力,可用于本文调查的HapMap数据库中11个人群和本文收集的8个人群的个体识别鉴定。     

广东省瑶族人群15个STR基因座的多态性调查

目的调查广东省瑶族人群无关个体 15个STR基因座 (D3S135 8、TH0 1、D2 1S11、D18S5 1、PentaE、D5S818、D13S317、D7S82 0、D16S5 39、CSF1PO、PentaD、vWA、D8S1179、TPOX、FGA)的多态性 ,研究其在法医学检验中的应用价值。方法应用PowerplexTM16荧光标记复合扩增系统对 2 2 2例广东省瑶族无关个体血样DNA进行 15个STR基因座的复合扩增 ,用ABI 310 0遗传分析仪对扩增产物进行检测 ,用GeneScan、GenoTyper软件进行基因分型 ,统计计算 15个STR基因座的群体遗传学参数。结果PowerplexTM16荧光标记系统的 15个STR基因座在广东省瑶族人群的累积偶合率为 7 5 8× 10 - 17,三联体累积非父排除率为 0 999998,二联体累积非父排除率为 0 9996 6。结论本研究 15个STR基因座可满足广东省瑶族人群法医学的个体识别及亲权鉴定的需要。     

3个miniSTR基因座四色荧光复合分型体系的构建及应用

目的建立D3S3053、D6S474、D20S482（扩增片段〈150bp）3个miniSTR基因座四色荧光复合分型体系,用于高度降解DNA样本的基因分型。方法采用6-FAM、HEX、TAMRA荧光染料标记D20S482、D3S3053、D6S474基因座上游引物,构建并优化复合扩增体系,在ABI310遗传分析仪上对扩增产物进行电泳分析,Genemapper3．2软件分析产物片段大小并进行分型。采用上述体系对120份河北汉族健康无关个体血样进行检测,并计算群体遗传学参数。比较该体系与ID试剂盒用于高度降解检材的成功率及灵敏度。结果D3S3053、D6S474、D20S4823个miniSTR基因座荧光标记复合扩增分型体系稳定可靠,灵敏度达50pg,用于高度降解检材分析的成功率明显高于ID试剂盒。3个基因座在河北汉族人群中的累积个人识别能力为0．998,累积非父排除率为0．84。结论该系统可用于分析高度降解DNA样本的基因分型,进行法医学个人识别和亲权鉴定。     

31个SNP位点多重PCR扩增和芯片分型技术的建立及其法医学应用

目的对SNP基因分型芯片在个体识别中的应用价值进行研究。方法根据SNP不同等位基因的序列设计探针,制成分型芯片。采用4个复合PCR体系,用末端标记了Cy5的引物进行复合PCR扩增,产物与寡核苷酸探针进行杂交,根据杂交产生的荧光信号值确定样品在各SNP位点的基因型。将这一方法应用于109份样本的分型,根据基因型分布统计分析31个SNP位点的法医学应用价值。同时,进行家系调查和方法灵敏度分析。结果方法的灵敏度为1ng;所检测的31个SNP位点的累积个体识别率为0.9999999999979(偶合率为2.13×10-12),二联体亲子鉴定中累积非父排除率为0.9609,三联体亲子鉴定中累积非父排除率为0.9970。家系调查的结果表明,这些位点等位基因由亲代向子代的传递符合孟德尔遗传定律。结论上述31个SNP位点为中高信息量位点,适用于法医学个体识别,可作为当前STR系统的补充。     

10个STR基因座荧光复合扩增体系的研制

目的建立10个STR基因座荧光标记复合扩增体系,并评价其法医学应用价值。方法在北京、山西、广东汉族,辽宁满族、西藏藏族群体中调查STR基因座遗传多态性,筛选出9个具有高度多态性和法医应用价值的STR基因座及性别基因座。构建四色荧光素标记复合扩增体系,制备等位基因分型标准物,编制分析软件,并对体系的种属特异性、灵敏度、稳定性、混合样本等检测能力进行考察。结果建立的复合扩增体系遗传稳定好,累积非父排除率可达0.999 96,累积个体识别率可达0.999 999 999 999 3;与CODIS系统均不存在连锁遗传;各基因座间布局合理、无杂峰、扩增结果清晰易辨,并可实现检测分析自动化。体系种属特异性较好,灵敏度为0.1ng,稳定性好,混合样本检出范围在2∶8～8∶2之间。实际案例检材检测结果好。结论本文建立的复合扩增体系在法医学实践中有较好的应用价值。     

SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot^®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10^?19 across all Western cat populations and the likelihood ratio was 1.52 × 10¹⁸. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.     

Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.     

基于等位基因特异性PCR原理建立的SNP分型新方法

目的建立一种新方法,对多个单核苷酸多态性(singlenucleotidepolymorphism,SNP)位点进行分型。方法基于等位基因特异性PCR原理,采用荧光标记复合扩增和毛细管电泳技术,根据PCR片段长度差异进行分型。选择SNP位点共11个,每个SNP位点设计两条长度不同、3’末端分别与SNP两个等位基因碱基配对的上游引物,同时为了增加特异性,在两条等位基因上游引物的3’末端第3或第4位碱基人为引入错配。在距离上游引物100～300bp范围内的合适位置,设计下游共用引物,并进行荧光标记。所有位点经过复合扩增后,PCR产物经ABIPrismTM310型遗传分析仪电泳分离,确定每个SNP的基因型。结果每个SNP位点纯合子为单一产物峰,杂合子则为长度不同的两个产物峰。不同的SNP位点扩增产物长度不同,根据产物长度和产物峰的数量进行SNP分型,一次完成11个SNP位点分型,其结果与直接测序完全一致。结论荧光标记复合扩增片段长度差异等位基因特异性PCR法是一种简单快速而有效的SNP分型新方法。     

Validating the consistency of cSNPs analysis results between DNA and RNA using SNaPshot method

The use of coding region single nucleotide polymorphisms (cSNPs) was recently proposed as a potential method for individual identification because it allows mRNA profiling and DNA typing to be performed concurrently. Nevertheless, availability of this approach still needs some further validation in different aspect. In this study, we have initially selected several SNP loci located in mRNA molecules that were confirmed to be highly expressed in blood. Both coding regions (CDRs) and untranslated regions (UTRs) were taken into consideration during the screening. Genomic DNA (gDNA) and total RNA from venous blood samples were isolated, followed by the synthesis of first-strand complementary DNA (cDNA) using purified RNA samples. Subsequently, the genotypes of these SNPs were respectively determined with gDNA and cDNA by using SNaPshot method. The PCR primers for cDNA were designed to span an intron in order to ensure that the amplification products were not due to the presence of potential DNA contamination. In summary, our study revealed a high consistency of cSNP analysis results between DNA and RNA on capillary electrophoresis platform, which highlighted the potential use of cSNP in forensic investigation.     

SNP分型方法及在法医学等领域的应用

随着单倍型图的产生,SNP越来越受到关注。不仅仅在Y染色体和线粒体,常染色体和X染色体上的SNPs的应用潜能也将被发现。SNPs具有比较低的突变率和适合于降解DNA分析的特点,在法医学领域也受到关注。本文综合介绍了SNPs和X-SNPs的一般特性、分型方法及其在法医学的应用。     

Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.     

A new approach to detect a set of SNP-SNP markers: Combining ARMS-PCR with SNaPshot technology

Microhaplotypes have become a new promising forensic genetic marker in recent years. The microhaplotype composed of two SNPs, SNP-SNP, indicates strong application potential because of the shortest fragment and good polymorphism and without the interference of stutter and high mutation rate as short tandem repeats (STR) and low polymorphism as a single SNP. Currently, the most common method to detect microhaplotypes is massively parallel sequencing (MPS), however its high cost and the need for special instruments limit its use in general forensic laboratories. In this study, we screened out 8 new SNP-SNP loci and established a new detection method by associating multiplex ARMS-PCR and SNaPshot technology. Firstly, we introduced ARMS-based PCR for SNP1. Then, SBE primers for SNaPshot assay were designed as 20–25 bp upstream complementary sequence next to the position of SNP2. Finally, 8 loci were built into one panel based on different SBE primer lengths and fluorescence colors. In brief, by combing ARMS-PCR and SNaPshot technology, it is easy and fast to profile the SNP1 and SNP2 orderly of the SNP-SNP microhaplotype based on CE platform. Our results suggested that the 8 loci have relatively high polymorphism as well as robust performance.     

H19基因上游差异性甲基化区SNPs多态性研究

目的获得H19基因上游差异性甲基化区中SNPs的群体遗传学信息。方法采用PCR和测序技术,对105例中国北方汉族健康无关个体H19上游启动子区检测;使用Haploview 4.1和PowerStats V12软件进行统计学分析。选用甲基化敏感的限制内切酶(msRE)HpaⅡ,检测5个家系样本H19等位基因的亲代来源。结果测序结果显示,H19启动子区含有13个SNPs,组成5种单倍型,13种单倍型组合,其个体识别能力为0.856、多态性信息含量为0.67、非父排除率为0.498。经msRE HpaⅡ消化母源等位基因后,进行PCR及测序分析,检测出父源等位基因,排除1例和肯定4例家系的亲缘关系。结论 DNA甲基化标记和SNPs多态性检测,可同时进行多态性分型并确定等位基因的亲代来源,具有较高的法医学应用价值。     

单核苷酸多态性研究进展

单核甘酸多态性（ SNPs）是继 RFLP和微卫星多态性标记之后的新一代遗传标记系统,具有密度高、遗传稳定、分析易自动化等特点。 SNPs可通过电泳、 PCR、酶切及测序等方法检测,已广泛应用于基因作图、疾病相关性分析、群体遗传学及药物研究等领域。     

常染色体21个SNPs多态性分型方法研究

目的建立常染色体21个SNPs的多态性分型方法。方法采用荧光标记公用引物和等位基因特异性引物原理设计SNP复合扩增引物体系,对45个备选SNP位点筛选,选出21个及性别Amelogenin构成复合扩增体系。PCR产物经3130XL型电泳仪电泳分离,GeneMaperTM3.0数据分析软件分析结果。同时随机选取6份样品,使用测序方法对SNP分型并进行测序验证。结果应用本研究建立的复合扩增体系扩增样品,产物经毛细管电泳后,每个SNPs均可正确判定基因型。随机选取6份样品SNPs位点测序结果显示,荧光标记SNPs复合扩增分型与直接测序结果完全一致。结论本研究建立的荧光标记公有引物特异性片段常染色体21个SNPs复合扩增方法是SNP多态性分析的一种有效方法,并有助于解决SNP分型识别能力、效率、通量和高成本的问题。