HLA-DRB1基因分型芯片的法医物证学应用价值研究

目的对HLA-DRB1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HLA-DRB1基因座不同等位基因的独特序列设计探针,制成分型芯片。将待测样品DNA用末端标记了CY5的引物进行PCR扩增,产物与芯片进行杂交,根据杂交产生的荧光信号值确定样品在HLA-DRB1位点的基因型。将这一方法应用于561份样本的HLA-DRB1基因分型,根据基因型分布统计分析其法医学应用价值。同时,进行了家系调查和方法灵敏度分析,并应用于部分案例。结果利用微量检材,HLA-DRB1基因芯片可检测DRB1位点等位基因26个,基因型的分布符合Hardy-Weinberg平衡定律,该位点的观察杂合度(Ho)为0.888,期望杂合度(He)为0.902,多态信息含量(PIC)为0.893,平均非父排除率(PE)为0.801。家系调查和案例运用的结果表明,HLA-DRB1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HLA-DRB1为高度多态位点,其基因分型芯片可在亲子鉴定和个体识别中发挥重要作用。     

HLA-DRBl基因分型芯片的法医物证学应用价值研究

目的对HLA-DRBl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HLA-DRBl基因座不同等位基因的独特序列设计探针．制成分型芯片。将待测样品DNA用末端标记了CY5的引物进行PCR扩增，产物与芯片进行杂交．根据杂交产生的荧光信号值确定样品在HLA-DRB1位点的基因型。将这一方法应用于561份样本的HLA-DFIBl基因分型，根据基因型分布统计分析其法医学应用价值。同时．进行了家系调查和方法灵敏度分析，并应用于部分案例。结果利用微量检材．HLA-DRB1基因芯片可检测DRB1位点等位基因26个，基因型的分布符合Harely—Weinberg平衡定律．该位点的观察杂合度(Ho)为0．888，期望杂合度(He)为0．902，多态信息含量(PIC)为0．893，平均非父排除率(PE)为0．80l。家系调查和案例运用的结果表明，HLA-DRB1位点等位基因由亲代向子代的传递符合盂德尔遗传定律。结论HLA-DRB1为高度多态位点，其基因分型芯片可在亲子鉴定和个体识别中发挥重要作用。     

基于二代测序平台的90个常染色体SNP分型研究

目的基于二代测序平台进行90个常染色体SNP位点分型,调查其在中国广东汉族人群中的多态性,评估其法医学应用价值。方法采集100例中国广东汉族无关个体外周血样,采用Auto Mate Express TM提取样本DNA,使用HID-Ion Ampli Seq?Identity Panel分型体系复合扩增90个SNP位点制备文库,Ion One Touch?2进行乳化PCR,Ion PGM?平台进行测序,Torrent_Suite_v4.4.2软件及HID_SNP_Genotyper_v4.3.1插件进行数据分析,计算常用法医学参数并与该群体Goldeneye TM 20A体系的检测效能进行比较。结果经Bonferroni法校正后,90个常染色体SNP位点分布均符合Hardy-Weinberg平衡,不存在连锁不平衡现象。各位点平均杂合度(Ho)为0.423,平均个体识别力(DP)为0.560,平均多态信息含量(PIC)为0.329。90个SNP体系的累积个体识别率(CDP)为(1-1.20×10~(-33)),大于20A体系;三联体累积非父排除率(CPE_(tri))为0.999 999 911,二联体累积非父排除率(CPE_(duo))为0.999 882,均小于20A。结论 90个常染色体SNP检测体系可独立应用于法医个体识别和三联体亲子鉴定,并辅助进行二联体亲子鉴定。     

HLA-A基因座多态性在亲子鉴定中的应用研究

首次将HLA A基因座的聚合酶链反应 寡核苷酸探针 (PCR SSOP)杂交分型技术应用于亲子鉴定案例分析 ,以获得HLA A基因座多态性用于法医学鉴定分析的基础数据。HLA A基因座基因分型的等位基因检出率 ( 2 4个 )和非父排除率 ( 73 3 % )均高于血清学检测方法 ;正常家系分析结果符合孟德尔遗传规律。对 3 9个亲子鉴定例的成员进行HLA A基因检测 ,9例排除 ( 2 3 1% ) ;未排除的 3 0例的亲子关系概率在 65 2 %～ 99 5 %之间。用PCR SSOP技术对HLA基因座基因分型不仅可以应用于亲子鉴定和法医学个人识别 ,还可以应用于器官移植配型及人类遗传学研究。     

运用二重PCR和DNA芯片技术检测ABO基因型

目的以玻片为载体,用寡核苷酸探针杂交技术检测ABO基因型。方法根据ABO基因座外显子6和外显子7的3个SNP点的序列分布特征设计4条寡核苷酸探针,制成分型芯片。将待测样品DNA用末端标记了Cy5的引物进行二重PCR扩增,产物与芯片上的探针进行杂交,根据杂交产生的荧光信号确定样品的ABO基因型。结果利用ABO芯片,可对血斑、毛发等微量检材进行ABO基因型检测。对115名汉族无关个体的调查表明,ABO基因型的分布符合Hardy-Weinberg平衡,等位基因杂合度观察值和期望值分别为0.591、0.616,多态信息含量为0.544,二联体和三联体非父排除率分别为0.188、0.334,个体识别能力为0.777。结论通过DNA芯片检测ABO基因型的技术适用于法医学样本,可满足高通量的检测需求。     

DNA芯片技术检测HLA-DRB1和ABO基因型

用DNA芯片技术检测HLA-DRB1-ABO基因型。根据HLA和ABO不同基因亚型的独特序列设计探针,制成分型芯片;待检测样品经PCR反应标记上荧光之后,与探针在芯片上进行杂交,通过对杂交产生的荧光信号值进行分析,确定样品DRB1位点和ABO位点的基因亚型。将这一方法应用于111份样本的HLA-DRB1-ABO基因分型并将部分样品进行基因测序。检测结果证明本实验研制的HLA-DR-ABO基因分型芯片可准确分辨出DRB1位点30个等位基因、ABO位点6种基因型。该方法分辨率高、特异性强、重复性好、操作简便,对比常规的PCR-SSP方法,HLA-DR-ABO基因芯片方法更为直观,并具有集成化优势,可以在一张芯片上同时检测HLA和ABO位点,并实现一张芯片多人份,不仅适用于法医学亲子鉴定和个体识别,亦可应用于移植配型、HLA相关疾病及人类遗传学研究。     

成都地区汉族人群D2S441位点的遗传多态性研究

为研究 STR位点 D2S441的遗传多态性,为法医学应用提供基础数据,应用 PCR及 PAG电泳技术对 260名成都地区汉族无关个体进行了调查,共检出 9个等位基因及 26种基因型,首次获得汉族群体频率分布 ,其等位基因片段大小范围为 131～ 155bp。该位点基因型频率分布符合 Hardy－ Weinberg平衡。家系调查证实了等位基因的传递遵循孟德尔遗传规律。其个人识别能力（ Dp）、杂合度（ H）、多态性信息含量（ PIC）和非父排除率（ PE）分别为 0.9084、 0.7885、 0.7390和 0.5778,表明该位点在法医学个人识别及亲子鉴定中具有较高的实用价值。     

vWFⅢ基因座扩增片段长度多态性及其法医学应用

研究短串联重复vWⅢ基因应扩增片段长度多态性及其法医学意义。应用STR－PCR分型法对200名中国辽宁汉族无关个体的短串联重复VWFⅢ的多态性进行调查。共检出8个等位基因,频率分布在0．003～0．333,基因长度范围为154～182bP,个人识别能力为0．921,非父排除率为0．581。群体调查证实该基因座符合Hardy－Weinberg平衡,家系调查结果表明其遗传符合孟德尔方式。该基因座可作为人类群体遗传学的一个有价值的多态性标记,可用于法医学个人识别及亲子鉴定。     

191名白山市汉族CODIS系统9个STR位点群体遗传学调查

目的　白山汉族CODIS系统 9个STR位点基因频率调查及其法医学应用 ;　方法　实验样本从 191位无相关白山市汉族个体获取 ,采用PCR复合扩增及 310遗传分析仪自动基因分型 ;　结果　这 9个STR位点均为高识别率位点 ,同重庆汉族人群群体遗传学数据比较显示有显著性差异 ;　结论　基因频率适合于白山汉族人群同一性概率及亲子鉴定概率计算 ;中国南北方汉族在个体识别及亲子鉴定概率计算时应采用本民族自己的等位基因频率。     

亲子鉴定DNA分型亲权指数的简化计算法

PCR扩增DNA片段长度多态性位点，如AmP－FLP、PCR－STR分型技术，在亲子鉴定中应用日益增加。由于被选择应用的DNA位点均具高杂合度、高非父排除率，以及PCR技术取材广泛、灵敏度高等特点，PCR－DNA分型有。逐步取代常规血型检测的趋势。PCR－DNA分型结果表明，杂合子个体呈2条片段，纯合于及条片段，等位基因为共显性遗传，位点均具有不连续基因频率分布。所以，亲权指数计算并不复杂。本文依据单位点DNA分型特发，根据Esse。M6ller计算理论，提出一套简明亲权指数计算公式。同时对父子单亲亲子鉴定的亲权指数计算公式也作…     

SNP genotyping by multiplex amplification and microarrays assay for forensic application

OBJECTIVE: Research on the application feasibility of SNP genotyping for forensic identification by microarrays. METHODS: Oligonucleotide microarrays which could detect 34 different SNPs were used. After hybridization and washing, the arrays were scanned and fluorescence intensities analyzed using Microarray software. Population studies on 34 SNP loci were carried out in a sample of 109 unrelated Chinese Han individuals using oligonucleotide microarrays for genotype detection. The method was also applied to cases. RESULTS: According to the results of population studies, no deviations from Hardy-Weinberg equilibrium could be found. Among the 34 loci, 3 SNPs were low informative, 4 were medium informative and 27 were high informative. The combination discrimination power (CDP) of the 31 optimal polymorphic SNPs was 0.9999999999979. The matching probability was 2.13 x 10(-12). The average exclusion probability in paternity testing for duos was 0.9609. The average exclusion probability in paternity testing for trios was 0.9970. CONCLUSION: The data and case application demonstrated that SNP typing by oligonucleotide probe microarrays was a useful technique for paternity testing and individual identification. Combined with the 28 SNPs loci distributed on HLA-DRB1 and ABO genes, the combination discrimination power (CDP) was 0.9999999999999910. The matching probability was 9.02 x 10(-15). The average exclusion probabilities in duos and in trios were 0.9894 and 0.9992, respectively. It may be concluded that the 59 SNPs loci yield the same power in forensic identification as CODIS STRs currently used.     

Selection of twenty-four highly informative SNP markers for human identification and paternity analysis in Koreans

A number of DNA marker types suitable for human identification and parentage testing have been developed, of which single nucleotide polymorphisms (SNPs) merit attention as they are abundant, genetically stable, and amenable to high-throughput automated analysis. In this regard, 24 highly informative SNP markers representing each 22 autosome and both sex chromosomes were selected, and the allele and genotype frequencies of these SNPs were determined in a group composed of 30 unrelated Koreans. Based on frequency data from this group, the estimated probability of identity (P(I)) and probability of paternity exclusion (P(E)) with 22 autosomal SNP loci were 1.905x10(-10) and 98.9%, respectively. The SNPs in this study offer a small but highly accurate database that will be an essential reference for SNP-based forensic application in the future.     

X染色体上高信息量SNP位点及其法医学价值

人类X染色体长154.8Mb,其上密布SNP位点,蕴涵着大量的信息。用于法医学鉴定的X-SNP标记多态性好、突变率低。本研究从Hapmap、NCBI的数据库中筛选了167个高信息量SNP位点,这些位点的等位基因在北京汉族人群中的分布频率均高于0.3,通过高通量、高灵敏度的检测方法可对各个X-SNP位点进行分型验证,通过正确的统计学分析可得到其法医学多态性参数。X-SNP位点具有一些常染色体遗传标记无法比拟的优点,作为常规STR基因座的补充,能用于解决特殊的亲子鉴定案,性别鉴定和混合斑鉴定。     

ABO genotyping by duplex amplification and oligonucleotide arrays assay

Objective

Research on the application feasibility of ABO genotyping for forensic identification by oligonucleotide arrays assay.

Methods

Oligonucleotide microarrays which detect three different SNPs in exon 6 and exon 7 for ABO genotyping were used. After hybridization wash, the arrays were scanned and fluorescence intensities were analyzed using microarray population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals oligonucleotide arrays for genotype detection. The method was also applied to cases.

Results

Technique could identify six genotypes of ABO system and the results of GeneChip analyses confirmed by PCR–RFLP. According to the results of population studies, no significant deviations Hardy–Weinberg equilibrium could be found. The observed heterozygosity (H-obs) was 0.591. Expected heterozygosity (H-exp) was 0.616. The polymorphic information content (PIC) was the average exclusion probability in paternity testing for duos (PE (1)) was 0.188. The average exclusion probability in paternity testing for trios (PE(2)) was 0.344. The discrimination power 0.777.

Conclusion

The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.     

Genome‐wide Screen for Individual Identification SNPs (IISNPs) and the Confirmation of Six of Them in Han Chinese with Pyrosequencing Technology*

Abstract: Single nucleotide polymorphisms (SNPs) offer promise to forensic DNA analysts, but it remains uncertain whether a panel of individual identification SNPs can be as informative as the Combined DNA Index System short tandem repeats. Based on the highly accurate and publicly available HapMap SNP database (r21a) and a minor allele frequency cutoff of ≥0.45, we completed a genome‐wide screen through 3,905,819 SNPs with internally modified computer programs and identified 1439 SNPs with high heterozygosity and low F_st values among four populations (Utah Caucasian, Han Chinese, Tokyo Japanese, and Nigerian Yoruba). Using pyrosequencing technology, we studied six loci in a relatively large group of samples to determine whether these loci were as informative as the HapMap data suggest. These SNPs performed as expected in the Han Chinese in terms of heterozygosity and F_st. The 1439 identified SNPs should provide a comprehensive and reliable set of loci for identity and relationship testing.     

A new 39-plex analysis method for SNPs including 15 blood group loci

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-M?ller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

67个X-SNP位点的分型检测和连锁不平衡检验

目的 筛选一组在中国汉族人群中具有法医学应用前景的X-SNP位点.方法 根据dbSNP和HapMap两个数据库提供的位点信息和频率数据从X染色体上筛选出67个候选X-SNP位点,采用多重PCR联合基质辅助激光解析电离飞行时间质谱技术检测中国汉族人群428名无关个体,获得67个候选X-SNP位点在中国汉族人群中的频率数据...     

法医SNP复合检测体系的构建及应用

目的构建48-SNP位点复合检测体系,用于个体识别、性别鉴定、ABO基因分型。方法采集225份无关个体样本(血斑及口腔拭子),18份案例样本(不同组织及体液斑),选择43个常染色体位点、4个ABO基因位点和1个性别鉴定位点,根据单碱基延伸技术通过GenomeLabTMSNPstream基因分型系统进行SNP分型;并检测体系灵敏度、同一个体不同组织同一性及模拟腐败检材。结果 48-SNP体系分型结果与测序结果的一致性为100%,最小DNA检出量为0.25ng,不同组织来源样本检测同一性很好;利用该体系检测225名无关汉族个体,所有位点均符合Hardy-Weinberg平衡,整个系统的随机匹配概率为9.4×10-18,累积非父排除率(CEP)为0.999 788,累积个体识别率大于0.999 999 999 999 999 99。结论本文48-SNP体系能同时进行个体识别、ABO基因分型和性别鉴定,可以作为现有STR检验体系的补充。