Genotyping of five short tandem repeat loci via triplex and duplex PCR

We have developed a triplex PCR method for D3S1359, HumTH01 and HumTPO tetranucleotide loci and a duplex PCR method for HumFES/FPS and HumvWA31A tetranucleotide loci using high resolution polyacrylamide gel electrophoresis and silver staining. The methods were evaluated for paternity testing and individual identification and allele frequencies at these loci are reported for 189–3387 unrelated individuals in the Finnish population. The D3S1359 locus, especially, was found to be a highly informative locus. Seventeen alleles were found in the D3S1359 locus with a highest observed allele frequency of 0.199, a high exclusion power (PE) in paternity testing (0.78) and a high observed heterozygosity (0.89). The combined PE for these five loci was 0.99.     

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.     

Matching and partially-matching DNA profiles

The DNA profiles of two individuals can have 0, 1, or 2 pairs of alleles that are the same at each locus. These events may be called mismatches, partial matches or matches, respectively, and they have probabilities that depend on the population proportions of alleles as well as the population structure parameter theta. The observed and expected numbers of pairs of individuals with various numbers of matching or partially matching loci in FBI and Australian databases are found to be in good agreement provided theta is set equal to some small value greater than zero. The likelihood ratios for two individuals having a specified degree of relationship versus being unrelated also depend on the numbers of matching and partially matching loci, but even unrelated pairs of individuals can have likelihood ratios that support hypotheses of relatedness. Matching probabilities allow predictions to be made for the sizes of databases that are expected to contain a pair of individuals with high numbers of matching loci. It is very likely that two individuals with at least 9 matching loci among the 13 CODIS loci have already been typed.     

应用Miseq FGx平台检测降解骨骼样本效果初探

目的评估Miseq FGx平台检测降解骨骼样本效果。方法对10例基于PCR-CE方法 STR分型结果不理想的降解骨骼样本用Miseq FGx平台检测,比较两个平台STR位点的检出结果和检出率。结果 10例降解骨骼样本在Mi Seq FGx平台上的STR位点检出率均高于PCR-CE方法,一些用PCR-CE方法没有检出的STR位点,很有可能在Miseq FGx平台中检出。同时,Miseq FGx平台测序还获得了X-STR、Y-STR、SNP等遗传标记的大量信息。结论 Mi Seq FGx平台检测降解骨骼样本有较好的应用价值。     

岫岩满族9个STR基因座遗传多态性

目的用复合荧光扩增体系调查辽宁鞍山岫岩满族无关个体D6S1043、D7S3048、D9S925、D11S2368、D14S608、D15S659、D17S1290、D20S470和GATA198805等9个STR基因座的遗传多态性。方法用本实验室构建的9个常染色体STR基因座荧光复合扩增体系．对辽宁鞍山岫岩满族252个...     

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。     

Genetic variation of 13 STR loci in the four endogamous tribal populations of Eastern India

We have analysed 13 autosomal STR loci in four endogamous tribal populations from two eastern states (Orissa and Nagaland) of India. The Gadaba, Kuvi Khond and Lotha Naga populations have not been analysed for microsatellite genetic variation previously. The allele frequencies for all loci are within the range observed in the geographical region and racial background, though some alleles showed greater variation. Departures from the Hardy-Weinberg equilibrium were tested by three methods and two loci (THO1 and TPOX) showed significant departures for all measures in Gadaba and Lotha Naga populations. The exclusion probability and discrimination probability were high for all analysed loci in all populations. There is no evidence for association of alleles among the STR loci studied. This allele frequency information will be useful for forensic, paternity and population genetic studies.     

New multiplexes for Europe-amendments and clarification of strategic development

Recently, the ENFSI/EDNAP groups issued advice on the design of the next generation of STR multiplexes in order to encourage standardisation within Europe. As the result of collaborative experimentation within the EDNAP group, we demonstrated that the low molecular weight STRs had substantial benefits to detect degraded samples. We subsequently recommended adoption of three new mini-STR loci to improve the success rate of degraded DNA markers, concurrent with the reduction in size of the existing STR markers in current use. This also improves the discriminating power of the system which is important to improve the power of national DNA databases. Subsequent discussions have occurred with manufacturers and members of the ENFSI/EDNAP groups. Because significant time and investment is required to develop new multiplexes of 13+ STR loci, manufacturers indicated that it would be preferable to adopt a staged approach. Two differing, but parallel strategies have now emerged. The first strategy employs a 13 STR loci multiplex incorporating three mini-STRs into the current multiplex test. The second strategy employs a multiplex of six high molecular weight STRs (in current use), modified to provide smaller amplicons combined with an additional two loci of high discriminating power. Eventually, the two strategies will converge to provide a single multiplex of 15 STR loci. The process will be guided by the ENFSI/EDNAP groups.     

Sequenced allelic ladders and population genetics of a new STR multiplex system

The advent of PCR technology and use of short tandem repeat (STR) loci improves throughput and reduces costs whilst a high level of discrimination can be achieved. A new system, comprising seven STRs, was developed to compliment the existing systems. This paper describes the preparation of allelic ladders of the most commonly observed alleles of a new STR multiplex system (third generation; TGM multiplex); all alleles have been sequenced. Meioses studies estimated a mutation rate of 0-0.4% across loci. Statistical independence was investigated by employing exact tests; chi(2)-tests and excess homozygosity tests. The results demonstrated that the allele proportions do not differ from those expected and that there was no consequential dependence between loci. The discriminating power of the system was examined using 295 Caucasian, 140 Afro-Caribbean and 212 Asian unrelated samples, and was found to be approximately 1 in 50 million, 1 in 85 million and 1 in 20 million for each of these groups, respectively.     

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.     

荧光标记复合扩增STR检测技术在法医学中的应用

本研究用荧光标记8个STR位点及性别位点,于同一试管同步进行扩增后,在PE377全自动测序仪于一个泳道内,成功地对各位点进行基因型分离检测.将此方法应用于30多起案件检验,均取得满意结果.     

High‐Throughput STR Analysis for DNA Database Using Direct PCR,

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high‐throughput and cost‐effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter‐loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time‐ and cost‐saving manner.     

Haplotype data for the 12 RM Y-STR loci in a Syrian population

Researches with RM Y-STRs have shown that these loci provide substantially higher haplotype diversity and haplotype discrimination capacity in worldwide populations when compared with the YSTRs commonly used in genetic forensics. The aim of this study was to develop an allelic frequency database for the Syrian population living in Turkey in order to obtain population data of 12 RM Y-STRs. A total of 80 unrelated males from the Syrian population living in Turkey were typed with 12 RM Y-STRs loci: DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS526a/b, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. The highest GD was observed for the locus DYF399S1 (0.91), followed by loci DYS449 (0.86) and DYS518 (0.83). RM Y-STR haplotype diversity was found 1.00 in these samples. Based on the results of this study, the RM YSTR loci showed remarkable haplotype resolution power in the Syrian population, high genetic diversity and, therefore, demonstrating their usefulness in forensic identification cases.     

Diversity of 17-locus Y-STR haplotypes in Upper (Southern) Egyptians

Seventeen Y-STR loci included in the AmpF?STR^® Yfiler™ PCR Amplification kit were typed in a population sample of 208 males from Upper (South) Egypt. Of 204 observed haplotypes, 200 were unique (96.6%) and 4 were found twice each. The 17 loci gave a discriminating power of 0.9998. DYS458 showed the highest diversity as a single-locus marker (h = 0.868) along with a high frequency of microvariants and new alleles (22% of the sample). Other loci revealed duplicated and null alleles. Comparative analysis with Y-STR datasets of relevant populations and submission of the haplotypes to the Y-STR Haplotype Reference Database (YHRD) were undertaken.     

An STR forensic typing system for genetic individualization of domestic cat (Felis catus) samples

A forensic genotyping panel of 11 tetranucleotide STR loci from the domestic cat was characterized and evaluated for genetic individualization of cat tissues. We first examined 49 candidate STR loci and their frequency assessment in domestic cat populations. The STR loci (3-4 base pair repeat motifs), mapped in the cat genome relative to 579 coding loci and 255 STR loci, are well distributed across the 18 feline autosomes. All loci exhibit Mendelian inheritance in a multi-generation pedigree. Eleven loci that were unlinked and were highly heterozygous in cat breeds were selected for a forensic panel. Heterozygosity values obtained for the independent loci, ranged from 0.60-0.82, while the average cat breed heterozygosity obtained for the 11 locus panel was 0.71 (range of 0.57-0.83). A small sample set of outbred domestic cats displayed a heterozygosity of 0.86 for the 11 locus panel. The power of discrimination of the panel is moderate to high in the cat breeds examined, with an average P(m) of 3.7E-06. The panel shows good potential for genetic individualization within outbred domestic cats with a P(m) of 5.31E-08. A multiplex protocol, designed for the co-amplification of the 11 loci and a gender-identifying locus, is species specific and robust, generating a product profile with as little as 0.125 nanograms of genomic DNA.     

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。     

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.     

犬11个STR基因座的遗传多态性

目的调查犬11个STR基因座的群体遗传多态性。方法应用自主构建的犬11个STR基因座（PEZ1、PEZ2、PEZ3、PEZ5、PEZ6、PEZ8、PEZ12、FH2010、FH2054、FH2132和FH2611）荧光复合扩增体系，扩增105只犬的样本，统计各基因座扩增结果，并分析其群体遗传参数。结果11个STR基因座的累积非父排除率和累积个体识别率分别为0．9330621和0．9999999．平均杂和度和平均多态信息含量分别为0．502和0．640。结论该11个犬STR基因座的遗传多态性较好，可以有效用于犬的个体识别和亲权鉴定。     

Brazilian population data on the polymerase chain reaction-based loci LDLR,GYPA, HBGG,D7S8, and Gc

Gene and genotype frequencies in relation to the low density lipoprotein receptor (LDLR), glycophorin A (GYPA), hemoglobin G gammaglobin (HBGG), D7S8, and group specific component (Gc) loci were determined in a sample of 344 unrelated individuals (250 whites and 94 mulattoes) living in the city of S?o Paulo, Brazil. DNA was extracted from 5 mL of peripheral blood obtained from each of the 344 volunteers by the salting-out procedure. Polymerase chain reaction and reverse dot-blot analysis were performed with the Amplitype PM PCR Amplification and Typing Kit (Polymarker Multiplex; Applied Biosystems, Foster City, CA) under conditions recommended by the manufacturer. Estimated allele frequencies in the white sample were in the usual range of that of other United States and European population groups. In any case, genotype distributions for these loci did not deviate significantly from Hardy-Weinberg equilibrium proportions. Only 1 marginally significant (0.01 < P < 0.05) association, between loci HBGG and Gc, was detected in our mulatto sample out of a total of 20 possible pairwise comparisons of the 5 loci for both data sets. Allele frequencies were significantly different (P < 0.001) at the HBGG and Gc loci when the white and mulatto samples were compared. Biologic relationship exclusion probabilities (test powers) were calculated for the data. A Brazilian database has thus been established for the loci LDLR, GYPA, HBGG, D7S8, and Gc, 5 polymerase chain reaction-based loci systems that have been shown to be a useful tool for biologic relationship identification and exclusion.     

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.