STR位点D19S253和D8S1179的法医学意义及应用研究

为评估STR位点D19S253和D8S1179的法医学应用价值，应用PCR和PAG垂直电泳技术对两位点的种属特异性，检测灵敏度，以及同一个体不同组织分型的同一性及不同基质和不同保存时间的斑痕分型等与法医应用有关的问题进行了研究，D19S253和D8S1179位点的检测灵敏度分别为0．25ng及0．5ng，同时两位点具有较高的种属特异性，同一性及较好重复性，且能够复合扩增，表明D19S253和D8S1179是法医学检案中较实用的两个STR标记。     

GeneMarker® HID: A reliable software tool for the analysis of forensic STR data

Abstract: GeneMarker^® HID was assessed as a software tool for the analysis of forensic short tandem repeat (STR) data and as a resource for analysis of custom STR multiplexes. The software is easy to learn and use, and includes design features that have the potential to reduce user fatigue. To illustrate reliability and accuracy, STR data from both single‐source and mixture profiles were analyzed and compared to profiles interpreted with another software package. A total of 1898 STR profiles representing 28,470 loci and more than 42,000 alleles were analyzed with 100% concordance. GeneMarker HID was also used to successfully analyze data generated from a custom STR multiplex, with simplified and rapid implementation. Finally, the impact of the user‐friendly design features of the software was assessed through a time scale study. The results suggest that laboratories can reduce the time required for data analysis by at least 25% when using GeneMarker HID.     

汉族12个STR位点群体遗传学调查

目的240个汉族无关个体12个STR位点基因频率调查及其法医学应用;方法采用12位点复合扩增及变性聚丙烯酰胺凝胶电泳基因分型;结果该系统12个STR基因位点在汉族人群中均为高识别率位点,特别适合于陈旧血痕检验;结论12位点STR-PCR复合扩增系统检测方法简便,经济实用,在法医个体识别和亲子鉴定中具有应用价值。     

Unleashing novel STRS via characterization of genome in a bottle reference samples

The higher level of multiplexing possible with current sequencing technologies encourages adoption of additional STR loci to aid in mixture interpretation [1]. However, characterization of these loci and orientation on the human genome is vital for interlaboratory comparability and databasing. Currently, when a laboratory publishes population data from a locus not previously characterized for forensic use, there is no robust way to verify the locus designation, repeat region format, and fidelity of target. To address this, we have evaluated short- and long-read sequence data generated for reference materials included in the Genome in a Bottle Consortium (GIAB) [2] with the goal of reporting STR sequences for loci which may be of interest to the forensic community. Initially, we have analyzed GIAB data using Marshfield sets of primers (published in [3]), targeting over 600 microsatellite loci with STRaitRazor 3.0 [4]. In the future, this approach can be expanded to include other loci of interest. High-confidence STR sequence data will be made publicly available via GenBank record creation within the STRSeq BioProject [5]. As the cell lines represented in GIAB reference materials are available for purchase, this STR dataset represents a robust method for researchers to confirm targeted loci.     

A 27‐Locus STR Assay to Meet All United States and European Law Enforcement Agency Standards,

Different national and international agencies have selected specific STR sets for forensic database use. To enhance database comparison across national and international borders, a 27‐locus multiplex system was developed comprising all 15 STR loci of the European standard set, the current 13 STR loci of the CODIS core, the proposed 22 STR loci of the expanded CODIS core, 4 additional commonly used STR loci, and the amelogenin locus. Development required iterative primer design to resolve primer‐related artifacts, amplicon sizing, and locus‐to‐locus balance issues. The 19.5‐min assay incorporated newly developed six‐dye chemistry analyzed using a novel microfluidic electrophoresis instrument capable of simultaneous detection and discrimination of 8 or more fluorescent dyes. The 27‐locus multiplex offers the potential for a new international STR standard permitting laboratories in any jurisdiction to use a single reaction to determine profiles for loci they typically generate plus an expanded common STR profiling set of global interest.     

A new triplex STR system without irregular alleles by silver staining and its potential application to forensic analysis

In order to increase the discriminating power of DNA analysis in forensic science, we devised a new triplex STR system using three novel STR loci we previously reported, D14S299 (wglc5), D15S233 (wgldl), and 9q2h2. We designated this system a CDH triplex system. The CDH triplex system showed a high discriminating power, especially in Caucasians. This system is composed of three STR loci showing only regular tetranucleotide repeat alleles. We easily enlarged the databases mainly of Japanese, using this system, and compared them with those of Caucasian and Chinese. This CDH triplex system therefore appears to be useful for forensic practice.     

Allele sequences of six new Y-STR loci and haplotypes in the Chinese Han population

Human chromosome Y-specific short tandem repeat (Y-specific STR) markers have useful properties for forensic applications. However, there is a need to develop more Y-specific STR markers, because the discriminating power of each STR locus is limited. In the present study, we describe our results on six new Y-specific STR markers that were initially located using sequence database information by Ayub et al. and were named DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439. Our studies focused on the analysis of the DNA sequence for each allele at all six Y-specific STR loci in order to understand their structures in the human genome and to construct human allelic ladders, which are necessary for forensic DNA typing. In addition, the haplotype distribution for all six analyzed loci was studied in a Chinese Han population sample. The results indicate that DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439 are useful Y-specific STR markers for forensic sciences.     

Allele frequencies of six miniSTR loci of three ethnic populations in Singapore

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR product sizes. This study investigated the allele frequency of six miniSTR loci, D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045, in three Singapore populations. All loci showed a moderate degree of polymorphism with observed heterozygosity >0.6 for all three populations. The allele frequencies, forensic parameters and heterozygosity comparison with other CODIS STR in similar populations are presented.     

Constructing universal multiplex PCR systems for comparative genotyping

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.     

基于重组质粒制备STR分型阳性参照物

目的基于重组质粒制备可用于校准法医STR分型的阳性参照物。方法以常用阳性参照物9948人类基因组DNA STR分型为依据,基于重组质粒构建包含CSF1PO、D7S820、TH01等40个常染色体位点,DYS391、DYS522、DYS385a/b等22个Y染色体位点以及性别判定基因座Amelogenin的STR分型阳性参照物。将重组质粒定量、稀释后等比例混合,分别应用于DNATyper~?19、DNATyper~?24、DNATyper~?Y、Amp F?STR~?Identifiler~?Plus以及Power Plex~?18D System五种扩增试剂盒。结果阳性参照物中各重组质粒浓度为0.01pg/μL~0.001pg/μL;应用于Amp F?STR~?Identifiler~?Plus PCR扩增试剂盒,基于重组质粒制备的阳性参照物与人类基因组DNA扩增检测结果差异较小;将此阳性参照物分别应用于不同公司、不同STR基因座的四种STR扩增试剂盒,电泳检测图谱显示各基因座基因型完整,分型正确,峰高相当,基因座间均衡性良好。结论基于重组质粒制备STR分型阳性参照物,是一种可以替代细胞系制备阳性参照物的方法,具有一定的参考价值。基于此方法制备的阳性参照物可适用于市面上常用的STR检验试剂盒,普适性较强,对法医DNA分型检测有一定的实用价值。     

Inferring recent human phylogenies using forensic STR technology

STR loci are characterized by extremely high mutation rates and thus, high levels of length polymorphism both within and among populations. In addition, much of the observed variation is believed to be nearly selectively neutral. Because of these features, STRs are ideal markers for genetic mapping, intra-species phylogenetic reconstructions and forensic analysis. In the present study, we investigate the application of five STR loci (CS1PO, TH01, TPOX, FGA and vWA) routinely used in forensic analysis for delineating the phylogenetic relationships of 10 human populations representing the three major racial groups (African-Caribbean, Croatian from the island of Hvar, East Asian, Han Chinese, Italian, Japanese, Portuguese, UK Caucasian, US Caucasian and Zimbabwe). The resulting tree topology exhibited strong geographic and racial partitioning consistent with that obtained with mtDNA haplotypes, Y-chromosome markers, SNPs, PAIs (polymorphic Alu insertions) as well as classic genetic polymorphisms. These findings suggest that forensic STR loci may be particularly powerful tools and provide the necessary fine resolution for the reconstruction of recent human evolutionary history.     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.     

Mutations at 17 STR loci in Chinese population

Knowledge about mutation rates and the mutational process of short-tandem-repeat (STR) or microsatellite loci used in forensic analysis is crucial for the correct interpretation of resulting genetic profiles. We analysed a total of 19,754 samples from 6532 paternity testing cases at 17 STR loci which are commonly applied to forensics. The parenthood in each of these cases was highly validated (probability>99.99%). We identified 178 mutations. Locus-specific mutation rate estimates varied between 7.0 x 10(-5) and 2.2 x 10(-3), and the overall average mutation rate estimate was 8.4 x 10(-4). The observed mutational features for STRs have important consequences for forensic application such as the definition of criterions for exclusion in paternity testing and the interpretation of DNA profiles in identification analysis. In order to enrich the reference data of STRs mutations which are valuable for forensic application, we suggest the establishment of such database and ask the whole forensic community for data contribution including China.     

Presentation of a three-banded allele pattern--analysis and interpretation

The Israel police forensic biology laboratory received as an item of evidence in an attempted murder case, a pair of trousers belonging to a suspect. A bloodstain was observed on the trousers and analyzed by STR typing for nine loci using the Promega GenePrint STR silver stain detection kits. The genetic profile defined was found to be identical to that of the victim's at all nine loci. Within this profile a three-banded allele pattern was observed at the D16S539 locus, both in the bloodstain and in the victim's reference blood sample. Confirmation of this phenomenon was accomplished by amplifying the extracted DNA from both the trousers and the victim's blood sample using the PowerPlex 16 kit by Promega and the AmpFlSTR SGM Plus kit by Perkin Elmer, followed by analysis of the amplification products by capillary electrophoresis on the ABI prism 310 genetic analyzer. The same three-banded allele pattern was observed at the D16S539 locus in both specimen and reference DNA, using each of the three kits. Three additional loci located on chromosome 16 (D16S3407, D16S2617 and D16S3082), not employed for forensic identification, were also analyzed and did not show three-banded allele pattern.     

Linear mixture analysis: a mathematical approach to resolving mixed DNA samples.

With the advent of PCR-based STR typing systems, mixed samples can be separated into their individual DNA profiles. Quantitative peak information can help in this analysis. However, despite such advances, forensic mixture analysis still remains a laborious art, with the high cost and effort often precluding timely reporting. We introduce here a new automated approach to resolving forensic DNA mixtures. Our linear mixture analysis (LMA) is a straightforward mathematical approach that can integrate all the quantitative PCR data into a single rapid computation. LMA has application to diverse mixture problems. As demonstrated here on laboratory STR data, LMA can assess the quality and utility of its solutions. Such rapid and robust methods for computer-based analysis of DNA mixtures may help in reducing crime.     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

New autosomal STR loci

Additional STR loci can be beneficial for a number of human identity, forensic casework, and DNA database applications. The marker selection and characterization process applied at NIST in developing these new loci and assays are described along with concordance testing results from non-overlapping PCR primers. A 23plex for simultaneous amplification of 22 autosomal STR loci and an amelogenin sex-typing assay is also demonstrated.     

浙江汉族人群6个STR基因座的遗传多态性的调查

目的进一步完善浙江省汉族人群STR基因座遗传多态性的调查,为其应用提供基础数据。方法采用AmpF1STRSGMplus和AmpF1STRCoficer反应试剂盒,使用ABI310型基因分析仪对浙江汉族人群200名无关个体血样进行了D16S539、D2S1338、D19S433、TH01、TPOX和CSF1PO6个STR基因座遗传学分析。结果分别发现了9、15、15、11、8、10个等位基因,发现的基因型分别为23、42、35、19、16、17个,其分布经X2检验均符合Hardy-Weinberg平衡定律,并分别统计了6个STR基因座的H、DP、PM、PE及PIC参数。结论6个STR基因座适合法医学应用。     

The First Successful Use of a Low Stringency Familial Match in a French Criminal Investigation

We describe how a very simple application of familial searching resolved a decade‐old, high‐profile rape/murder in France. This was the first use of familial searching in a criminal case using the French STR DNA database, which contains approximately 1,800,000 profiles. When an unknown forensic profile (18 loci) was searched against the French arrestee/offender database using CODIS configured for a low stringency search, a single low stringency match was identified. This profile was attributed to the father of the man suspected to be the source of the semen recovered from the murder victim Elodie Kulik. The identification was confirmed using Y‐chromosome DNA from the putative father, an STR profile from the mother, and finally a tissue sample from the exhumed body of the man who left the semen. Because of this identification, the investigators are now pursuing possible co‐conspirators.     

武汉汉族人群D20S85和D6S477基因座遗传多态性调查

目的对武汉地区280名汉族无关个体的D20S85和D6S477位点遗传多态性进行调查,研究其在法医学检验中的应用价值。方法应用PCR和PAGE技术进行分型检验。结果分别检出10个、9个等位基因,获得各等位基因在该地区汉族人群分布频率,基因型频率分布符合Hardy-Weinberg平衡。两位点的DP值分别为0.9085、0.9127。结论D20S85和D6S477是法医学中重要的遗传标记。