Testing and evaluation of 43 "noncore" Y chromosome markers for forensic casework applications

A developmental validation study was performed on three Y-STR multiplex systems, Multiplex III (MPIII), Multiplex IV (MPIV), and Multiplex V (MPV), to ascertain their potential applicability to forensic casework. MPIII contains eight Y-STRs, including DYS426, DYS435, DYS436, DYS441, DYS442, DYS446, DYS462, and Y-GATA-A10, and one InDel, YAP (DYS287). MPIV contains 21 Y-STR loci, including DYS443, DYS444, DYS445, DYS447, DYS448, DYS449, DYS452, DYS453, DYS454, DYS455, DYS456, DYS458, DYS463, DYS464, DYS468, DYS484, DYS522, DYS527, DYS531 DYS557, and DYS588. MPV contains 13 Y-STR loci, including DYS459, DYS476, DYS488, DYS513, DYS549, DYS561, DYS570, DYS575, DYS576, DYS590, DYS594, DYS598, and DYS607. Full genetic profiles were consistently obtained for all three multiplexes with 25-50 pg of male DNA. No significant amplification was observed with 1 mug of female DNA. Each multiplex permitted the determination of the number of male donors in male:male DNA admixtures. Species specificity studies demonstrated some cross-reactivity with some primate samples. Environmentally compromised blood samples produced full or partial profiles after exposure to various conditions for up to 1 year. Full profiles were recovered from simulated casework specimens including cigarette butts and postcoital cervicovaginal swabs. Population data were collected to determine individual loci gene diversity and multiplex discriminatory capacity.     

Forensic application of Y chromosome SNPs in inconclusive cases

Biallelic markers, Single Nucleotide Polymorphisms (SNPs), are nowadays a powerful tool in the analysis of degraded samples. Namely, Y chromosome SNPs allow to determine the gender of the analyzed sample and to establish its haplogroup, making possible to attribute the ethnicity of male individuals. The aim of this study is to obtain Y-SNPs in forensic samples without STRs results, checking methodologies previously used.     

The use of the LightCycler for the detection of Y chromosome SNPs

A novel methodology based on PCR monitoring on-line with fluorescent formats using the LightCycler for Y chromosome SNP typing is proposed. The main advantages of the system are the time necessary for the analysis (which is around 20 min), the robustness and the accuracy of the method and especially its sensitivity, which permits the detection of the male component in male-female mixtures up to 1:300 for some of the SNPs.Singleplexes of four different SNPs (M9, sY81, SRY-1532 and SRY-2627) as well as two duplexes (M9 and sY81 on the one hand and SRY-1532 and SRY-2627 on the other) were efficiently implemented. A simultaneous amplification and analysis of the four SNPs is also possible. It seems difficult with the current methodology to implement more than a quadruplex.     

Population structure of Y chromosome SNP haplogroups in the United States and forensic implications for constructing Y chromosome STR databases

A set of 61 Y chromosome single-nucleotide-polymorphisms (Y-SNPs) is typed in a sample of 2517 individuals from 38 populations to infer the geographic origins of Y chromosomes in the United States and to test for paternal admixture among African-, European-, Hispanic-, Asian-, and Native-Americans. All of the samples were previously typed with the 11 core U.S. Y chromosome short tandem repeats (Y-STRs) recommended by SWGDAM, which revealed high levels of among ethnic group variation and low levels of among-population-within-ethnic-group variation. Admixture estimates vary greatly among populations and ethnic groups. The frequencies of non-European (3.4%) and non-Asian (4.5%) Y chromosomes are generally low in European-American and Asian-American populations, respectively. The frequencies of European Y chromosomes in Native-American populations range widely (i.e., 7-89%) and follow a West to East gradient, whereas they are relatively consistent in African-American populations (26.4+/-8.9%) from different locations. The European (77.8+/-9.3%) and Native-American (13.7+/-7.4%) components of the Hispanic paternal gene pool are also relatively constant among geographic regions; however, the African contribution is much higher in the Northeast (10.5+/-6.4%) than in the Southwest (1.5+/-0.9%) or Midwest (0%). To test for the effects of inter-ethnic admixture on the structure of Y-STR diversity in the U.S., we perform subtraction analyses in which Y chromosomes inferred to be admixed by Y-SNP analysis are removed from the database and pairwise population differentiation tests are implemented on the remaining Y-STR haplotypes. Results show that low levels of heterogeneity previously observed between pairs of Hispanic-American populations disappear when African-derived chromosomes are removed from the analysis. This is not the case for an unusual sample of European-Americans from New York City when its African-derived chromosomes are removed, or for Native-American populations when European-derived chromosomes are removed. We infer that both inter-ethnic admixture and population structure in ancestral source populations may contribute to fine scale Y-STR heterogeneity within U.S. ethnic groups.     

Validation and casework application of a Y chromosome specific STR multiplex

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor’s alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.     

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.     

HLA区域SNPs及其在法医学中的应用价值

人类白细胞抗原是迄今为止人类发现的最复杂的基因系统。HLA区域SNPs的分布频率高于人类整个基因组水平达到8.6%。目前SNPs可用多种方法检测。本文主要综述HLA区域SNPs在法医学亲权鉴定和个人识别的应用价值。     

Frequency assessment of SNPs for forensic identification in different populations

Allele frequencies for 16 previously described autosomal SNPs were tested in 1020 unrelated individuals originating from three different continents (Africa, Asia and Europe). The populations analyzed included Africans from Benin Gulf (180), Asians from Mongolia (160) and Europeans from Italy (680).     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

Performance evaluation and optimization of multiplex PCRs for the highly discriminating OSU 10-locus set Y-STRs

In a previous study, a new set of Y-chromosome short tandem repeats, the OSU 10-locus set (MPM1 and MPM2), was shown to have a higher discrimination power when evaluated against the 10 SWGDAM loci on a common population panel. Here, we describe the optimization of the multiplex reactions using dye-labeled primers followed by performance evaluations. The loci exhibited high precision, human male specificity, reliability in different body fluids, high sensitivity, stability, and the ability to amplify nonprobative casework and mixture samples. Stutter for the all of the loci, with the exception of the highly polymorphic locus DYS688, was similar to that observed for autosomal loci. The results of the performance evaluations reinforced the utility of these loci.     

Y chromosome J2 subtyping in an Italian sample: Population and forensic implications

In the recent years the Y chromosome genealogy has been refined by a number of newly discovered SNPs. The non-random distribution of the Y chromosome lineages worldwide makes fundamental the dissection and characterisation of haplogroups associated with specific geographic areas. In Southern Europe the haplogroup J2, as defined by the M172 marker, can reach frequencies up to 35%, making the dissection of such lineage critical for population studies. Here we present a study on J2 chromosomes from the Italian peninsula. Populations and forensic implications are discussed. A total of 900 individuals were previously genotyped for a number of SNPs, including M172. More than 200 of these have been now genotyped for 7 SNPs within the J2 lineage using a multiplex SNaPshot approach. The different distribution of the various lineages in different geographic areas probably reflects different historical demographic events and points to differential Y chromosome haplotype distribution, with implication for forensic application of this genetic marker.     

A PCR multiplex and database for forensic DNA identification of dogs

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.     

microRNAs及其法医学应用前景

microRNAs(miRNAs)是一类内源性的非编码小分子RNA,平均长度约22nt,广泛存在于各种真核细胞中,并参与调节细胞生长发育、分化、凋亡、衰老、疾病及肿瘤的发生等众多重要生命活动。基于其生物学功能,miRNAs可能在法医学体液来源鉴定、个体年龄推断、同卵双生子甄别等方面具有广阔的应用前景。     

荧光STR直接复合扩增试剂缓冲体系的研制

目的研制适用于数据库样本荧光STR直接复合扩增体系。方法针对常规血卡、FTA和903血卡样本,配制扩增缓冲液基准母液,采用不同配方的扩增缓冲体系进行直接扩增及检测。考察不同种类增强剂、4种商业化DNA聚合酶、不同复性温度和终延伸时间对检材的检测效果,并验证优化体系的适应性。结果采用本文所建体系对各类血卡样本进行检验,均可获得样本清晰、完整的STR分型。体系选择BSA\Tween20\DMSO\甘油等增强剂组合、Typer热启动聚合酶1.5U/10μL、57～59℃复性温度、30～50min终延伸时间,采用10μL体系即可对直径1.2mm FTA卡血样进行有效分型。结论本文所研制的缓冲体系能够满足常规血卡、FTA和903血卡样本直接扩增检验的需要。     

Possible applications of Y chromosome STRs in examination of abortion tissue

By application of Y-chromosomal STRs, DNA analysis of abortion material can be considerably facilitated since great excess of maternal DNA is tolerated without disturbing the Y-STR amplification. If paternity can't be excluded on the basis of the Y-STR haplotype, further examinations must follow, e.g. autosomal STR analysis. For this purpose, histological preparation of the abortion tissue might still be necessary. Different Y-chromosomal haplotypes of embryo and putative father usually lead to an exclusion from paternity. Based on four case examples, the feasibility of this method is discussed.     

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.     

Y－染色体SNPs及其在法医学中的应用价值

随着人类基因组计划的迅猛发展,已有越来越多的Y染色体SNP位点被发现,在个人识别、家系谱的建立、疾病的预测与诊断方面,Y染色体单核苷酸多态性提供了非常有价值的遗传标记。同样在法医学中也有广阔的应用前景。本文综合介绍了SNP和Y-SNP的一般特性及在法医学中的应用价值。     

Y chromosome SNP analysis in a Portuguese population sample using a multiplex PCR and minisequencing strategy

The study of single nucleotide polymorphisms (SNPs) located on the Y chromosome specific region Y-SNPs (92R7, M70, M22, Tat, P25, SRY10831, M173, M213 and M9) was used to characterize a population sample from Central Portugal, in order to investigate the frequency distribution of the male lineages and to compare the observed results with those obtained in other Portuguese regions.The genotyping strategy was according to the described by Brion et al. [M. Brion, et al., Int. J. Legal Med. 119 (2004) 10-15].In this population sample from Central Portugal a typical Western European haplogroup composition was found. The majority of samples (almost 70%) were assigned inside haplogroup R. As for other Iberian populations, the most frequent haplogroup was R1b-P25 (52.2%), followed by F(xK)-M213 (15.2%), E-B-SRY10831.1, R1(xR1a,b)-M173 and R1a-SRY10831.2 (each of them with a 8.7% frequency), K2-M70 (4.3%) and L-M22 (2.2%). When comparing our sample with other samples from Portugal, no significant differences were found.     

线粒体DNA的研究进展及其法医学应用

线粒体DNA(mitochondrialDNA,mtDNA)是存在于细胞质内的环状DNA。它的存在早在三十多年前就有人提出。如今,关于线粒体的研究领域是生物医学中发展最快的学科之一。它的发展基于一些很基本且有趣的问题的提出,这些问题主要是关于线粒体是如何进化,如何产生能量。另外,在疾病中线粒体基因如何发生突变、细胞凋亡如何受到它的调节、以及衰老如何对线粒体DNA发生影响等问题都有待解答,而且对这些问题的探讨将会对诸如人类学、法医学以及疾病的治疗有很大的用途。     

孔姓人群Y染色体遗传多态性研究及其法医学意义

目的获取孔姓人群Y-SNP和Y-STR遗传标记的信息,探索姓氏与Y染色体的关联性及法医学意义。方法采用等级复合扩增和SNaPshot技术检测255例孔姓男性和330例随机无关男性样本的12个Y-SNPs位点信息;采用Y filer试剂盒检测孔姓人群的17个Y-STRs基因座;应用Arlequin 3.5.1.2、Network4.6.1.1进行统计分析。结果12个Y-SNPs位点构成13种单倍群分型,孔姓人群和无关人群中最高分布频率的单倍群均为O3a2c1a-M117(21.57%,14.85%)。孔姓人群17个Y-STRs基因座构成的196种单倍型,多态性为0.993 9,单倍型14-12-25-28-19-15-12-19-12-11-12-22-12-11-14-10-19出现15次。O3-M122单倍群的中介网络树及不配对差异分析显示孔姓人群有两个中心星型分布,核苷酸不配对曲线呈单峰泊松分布。结论联合Y-SNP和Y-STR遗传标记分析表明孔姓人群存在复杂的基因交流,有多个姓氏起源,且在历史上经历过一定的扩张或持续增长,结合孔姓家谱历史分析其人群结构的遗传差异在法医学方面有潜在的应用价值。