AmpFlSTR MiniFiler™ PCR amplification kit: The new miniSTR multiplex kit

The AmpFlSTR^® MiniFiler™ PCR amplification kit (Applied Biosystems), a new available 8-miniSTR and the sex determining marker Amelogenin multiplex, includes the most common problematic loci (above 200 bp) of the AmpFlSTR^® Identifiler™ PCR amplification kit: FGA, D21S11, D18S51, D13S317, D7S820, D16S539, CSF1PO and D2S1338.Several casework samples with different DNA contents were tested.Results allowed to complete partial Identifiler™ profiles and additional information was achieved in low copy number (LCN) samples, revealing that this miniSTR kit can improve identification of compromised samples.     

Developmental validation of the PowerPlex ESX 16 and PowerPlex ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex^® ESX 16 (European Standard Extended 16) and the PowerPlex^® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex^® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR^® SGM Plus^® kit as standard size. The PowerPlex^® ESX 17 System amplifies the same loci as the PowerPlex^® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52-95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

The Upper Silesia (Poland) population and forensic usefulness of 15 autosomal STR loci

Allele frequencies, forensic parameters for the 15 STR loci in the AmpFlSTR^® Identifiler Kit (Applied Biosystems), D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D19S433, VWA, TPOX, D18S51, D5S818 and FGA were determined in a sample of 150 unrelated dead and alive adults from the Upper Silesia region (Poland). The values of heterozygosity (Ht), polymorphic information content (PIC), power of discrimination (PD), matching probability (PM), mean exclusion chance (MEC) and mean exclusion probability (MEP) were calculated. Possible divergence from HWE was determined. Comparison of allele frequencies for examined STR loci between the Upper Silesia population and other Polish populations was carried out.     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

STRs data for the loci D3S1385, vWA,FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 from Córdoba (Argentina)

Allele and genotype frequencies for nine STRs loci included in the AmpFlSTR Profiler Plus kit (D3S1385, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820), were determined from urban and countryside population of Córdoba (Argentina). All loci meet the Hardy-Weinberg expectation, and there is little evidence for alleles association between these nine loci. The results demonstrate that these loci can be useful for databasing purposes in human identification and parentage testing in the population of Córdoba (Argentina).     

Genetic polymorphisms of 20 STR loci in Chinese Han population in Tianjin,North China

The PowerPlex^® 21 System PCR Amplification Kit was a new PCR Amplification Kit developed for forensic laboratories, but there was a lack of data about this kit in Chinese people in Tianjin, North China. This kit contained 20 STR loci, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA. In order to evaluate this kit and to get basic population data for its use in forensic practice in Chinese Han population, 360 unrelated Chinese Han individuals from Tianjin were typed using the Kit. Allele frequencies of the 20 STR loci and further population forensic genetic parameters were obtained. The observed genotype frequencies and expected genotype frequencies were evaluated by χ² test. No significant deviation from the Hardy–Weinberg equilibrium was observed in the population sample for the 20 STR loci. The population data in the present study can be used for routine forensic practice in Tianjin, North China.     

青岛地区汉族人群13个STR基因座的频率分布及法医学应用

目的　调查青岛地区汉族人群无关个体的 13个STR基因座 (D3S135 8、VWA、FGA、D8S1179、D2 1S11、D18S5 1、D5S818、D13S317、D7S82 0、D16S5 39、TH0 1、TPOX、CSFIPO)的基因频率分布 ,研究其遗传多态性及其在法医学个体识别及亲子鉴定中的应用价值。　方法　用美国ABI - 310型遗传分析仪对ProfilerPlus和Cofiler两个系统的 13个STR基因座的复合扩增产物进行毛细管电泳及四色荧光自动分析检测 ,基因分型软件为GeneScanv3.1和Genotyperv2 .5 .2。　 结果　获得 13个STR基因座在青岛地区汉族人群的基因频率分布数据 ,13个STR基因座的PIC >0 .5 ,DP >0 .71,CCE =0 .999999,TDP值接近 1,TPm =1.2× 10 -14 ,家系调查符合孟德尔遗传规律。　结论　ProfilerPlus和Cofiler两个系统的 13个STR基因座在法医学个体识别及亲子鉴定中具有较高的应用价值。     

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.     

6个miniSTR基因座荧光标记复合扩增的遗传多态性

目的评价6个miniSTR基因座在DNA高度降解检材中的法医学应用价值,并调查广东汉族人群6个miniSTR基因座的遗传多态性。方法采用两个复合扩增PCR体系、四色荧光标记及毛细管电泳技术,对D1S1677,D2S441,D4S2364,D10S1248,D14S1434,D22S1045基因座进行基因型检测。结果6个miniSTR基因座均获得了清晰的基因型分型结果,扩增片段均小于120bp,分别检出7、7、5、8、8、7个等位基因和14、11、11、19、12、14种基因型,基因型分布均符合Hardy-Weinberg平衡。6个miniSTR基因座在广东汉族群体的个人识别率和非父排除率分别依次为0.863、0.895、0.792、0.894、0.814、0.904和0.392、0.360、0.353、0.568、0.378、0.513。10例IdentifilerTM试剂盒未能正确分型的高度降解DNA样本,采用6个miniSTR基因座复合扩增体系检测均提高了分型成功率结论6个miniSTR基因座荧光标记复合扩增体系在DNA高度降解检材的检测中具有较高的应用价值,并且在广东地区汉族群体中具有较好的遗传多态性。     

Fluorescent multiplex analysis of nine STR loci: Spanish population data

A total of 171 Caucasians living in Andalucia (southern Spain) have been typed for nine short tandem repeat (STR) loci by multiplex PCR amplification using a commercially available kit (Profiler Plus; Perkin–Elmer, Norwalk, CT, USA) and semi-automatic electrophoresis (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The kit enables typing of the STR loci D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, and D18S51. All loci, except D7S820, meet Hardy–Weinberg equilibrium. Because of the large number of loci that can be analyzed, the power of discrimination (PD) is greater than 0.99999, and the probability of exclusion (PE) reaches 0.99991 in our population sample.     

甘肃省汉族人群18个STR位点的遗传多态性

目的 调查18个短串联重复序列（Short Tandem Repeat,STR）位点在甘肃省汉族人群中的基因频率分布.方法 采用PCR扩增及毛细管电泳技术对272名个体的18个STR基因座进行分析.结果 共检出202种等位基因,基因频率分布在0.002～0.570之间.18个STR基因型分布均符合Hardy-Weinberg平衡（P＞0.05）,杂合度在0.599～0.893之间,个人识别能力在0.771～0.984,多态信息含量在0.534～0.910,非父排除概率在0.290～0.782.结论 本研究结果可为人类群体遗传学及法医学后续研究提供详实可靠的基础数据.     

Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.     

Effect of low-dose radiation on mutation rates of STR loci commonly used in forensic casework

The possible effect of low-dose radiation on STR markers in people exposed to radiation during their professional activities was estimated in this study. We evaluated mutation rates in 17 forensic STR loci typed by the COrDIS-18 kit (CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D21S11, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, and vWA) in 78 families (father–mother–child) with one parent exposed to low-dose radiation before fertilization. Five mutations were observed. In two cases, the new alleles were delivered from the non-exposed parent. The calculated mutation rate for the 17 studied STR loci in families appeared to be in good concordance with data published for normal populations. No evidence for an elevated mutation rate in STR markers after low-dose radiation was found.     

Population genetics of 15 autosomal STR loci in the population of Pomorze Zachodnie (NW Poland)

Allele frequency data and forensic efficiency parameters for 15 STR loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA were estimated from a sample of 600 unrelated individuals from the Pomorze Zachodnie (NW Poland). The combined MP and PE for all 15 loci are 3.9 × 10⁻¹⁸ and 0.9999988, respectively. Pairwise comparisons between Northwestern Poland and other Polish populations were performed.     

Genetic analysis of 15 STR loci in the population of Zhejiang Province (Southeast China)

Fifteen autosomal STR loci were analyzed from a population sample of 598 unrelated individuals residing in Zhejiang Province. We report allele frequencies distribution and statistical parameters for all 15 STR loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA. Allele frequencies, the observed heterozygosity (Ho), the polymorphic information content (PIC), and the probability of paternity exclusion (PE) were calculated. All loci were in accordance with Hardy–Weinberg equilibrium (P > 0.05). Our studied population data were compared with the previously published population data of other ethnic groups or areas in China. Our results of present study were valuable for human identification and paternity tests in Zhejiang Province.     

The Upper Silesia (Poland) population and forensic usefulness of 11 autosomal STR loci

Allele frequencies, forensic parameters for the 11 STR loci in GenePrint STR Systems—Silver Stain Detection (Promega): CSF1PO, TPOX, TH01, D16S539, D7S820, D13S317, F13A01, FESFPS, VWA, F13B, LPL were determined in a sample of 529–1436 unrelated, dead and alive adults from the Upper Silesia region (Poland). Furthermore, gender identification was carried out. The values of heterozygosity (Ht), polymorphic information content (PIC), power of discrimination (PD), matching probability (PM), mean exclusion chance (MEC) and mean exclusion probability (MEP) were calculated using Dudek's computer programme (FatRec, High Technical School of Cz?stochowa, Poland). A possible divergence from HWE was determined by χ² and exact tests using Miller's computer programme (TFPGA, Northern Arizona University, Flagstaff, USA). Comparison of allele frequencies between the Upper Silesia population and other Polish populations was performed with Carmody's test.     

Validation of the AmpF?STR SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Validation of the AmpF?STR^® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpF?STR^® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.     

Automated extraction of DNA and PCR setup using a Tecan Freedom EVO liquid handler

We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO^® liquid handler mounted with a Te-MagS™ magnetic separation device. The DNA was extracted using the Qiagen MagAttract^® DNA Mini M48 kit. The DNA was amplified using AmpF?STR^® Identifiler^®, Y-filer^® (Applied Biosystems), GenePrint^® FFFL and PowerPlex^® Y (Promega). The methods were validated for fresh whole blood and blood from deceased according to EN/ISO 17025.     

中国北方汉族与维吾尔族群体8个STR位点的遗传多态性

目的调查中国北方汉族与维吾尔族群体8个STR位点的遗传多态性。方法应用荧光标记引物试剂盒及基因扫描技术检测vWA、TH01、TPOX、CSF1PO、D5S818、D13S317、D7S820、D16S539位点等位基因。结果100例汉族群体共检出62个等位基因,累计非父排除率为0.9975;50例维吾尔族群体共检出52个等位基因,累计非父排除率为0.9973。2群体总个人识别机率均超过0.9999。基因频率分布2群体间存在显著性差异。结论8个STR位点在汉族与维吾尔族群体中具有较高的遗传多态性,频率分布有民族差别。