Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

16个线粒体DNASNP复合检测体系的建立

目的建立一种复合检测线粒体DNA（mtDNA）单核苷酸多态性（SNP）分型的方法。方法通过设计等位基因特异性引物并结合毛细管电泳分型技术平台,建立包含16个mtDNASNP位点的复合扩增检测体系。对50名汉族无关个体血样进行mtDNASNP检测,并通过直接测序法对其分型结果进行验证。结果50个样本经本检测体系复合扩增后,均得到清晰的SNP分型结果,当模板量在0．5～10pg时能得到较理想的分型图谱。样本的复合检测结果与直接测序法结果完全一致。结论建立的复合检测体系检测mtDNASNP方法灵敏度高、操作简便、分型准确,为针对mtDNA进行简便、有效的中高通量多态性分型提供了一种新方法。     

Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.     

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.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

A 21‐Locus Autosomal SNP Multiplex and its Application in Forensic Science

To develop a cost‐effective technique for single‐nucleotide polymorphism (SNP) genotyping and improve the efficiency to analyze degraded DNA, we have established a novel multiplex system including 21‐locus autosomal SNPs and amelogenin locus, which was based on allele‐specific amplification (ASA) and universal reporter primers (URP). The target amplicons for each of the 21 SNPs arranged from 63 base pair (bp) to 192 bp. The system was tested in 539 samples from three ethnic groups (Han, Mongolian, and Zhuang population) in China, and the total power of discrimination (TPD) and cumulative probability of exclusion (CPE) were more than 0.99999999 and 0.98, respectively. The system was further validated with forensic samples and full profiles could be achieved from degraded DNA and 63 case‐type samples. In summary, the multiplex system offers an effective technique for individual identification of forensic samples and is much more efficient in the analysis of degraded DNA compared with standard STR typing.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

6FAM-HEX-TMR-ROX四色荧光分析体系的构建

目的为在310基因分析仪上进行6FAM-HEX-TMR-ROX四色荧光STR复合扩增分型建立基础条件。方法以pGEM载体作为靶DNA,设计引物扩增获得500~80bp的13个长度不等DNA片段,以这些片段纯化物作为模板DNA,分别扩增获得6FAM、HEX、TMR和ROX标记的M atrix标准物,用4种M atrix标准物在310基因分析仪上构建可用于6FAM-HEX-TMR-ROX四色荧光分析的M atrix文件。结果扩增所得的13个非标记片段,长度分别为80、100、120、140、160、180、200、220、260、300、340、400、500bp,在非变性聚丙烯酰胺凝胶连续电泳-银染凝胶上均表现为单条带,6FAM、HEX、TMR、ROX分别标记的片段通过310基因分析仪检测均为单峰。混合ROX标记的80、120、180、220、260、300bp的6个片段获得的内标,显示出良好的线性关系。结论建立了有效的6FAM-HEX-TMR-ROX四色荧光分析M atrix,为进行多个STR基因座荧光标记复合扩增检测奠定了基础。     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR^®PCR Amplification kits.     

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.     

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.     

Development of two new Mini-STR multiplex assay for typing archival Bouin's fluid-fixed paraffin-embedded tissues

Short amplicon autosomal short tandem repeat (Mini-STR) assay has proved to be a highly useful tool in forensic applications, especially for highly degraded DNA samples that typically result in partial profiles and total loss of information from regular STR amplicons.In this study two new quadruplex systems were designed to get nuclear DNA profile from degraded forensic casework samples. In order to obtain PCR products less than 120 bp in size, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned and assembled in two PCR-multiplexes: D8S1179, D3S1358, TPOX, D16S539 and CSF1P0, TH01, D13S317, D5S818.After validation, these two Mini-STR quadruplex were employed in paternity testing case that involved DNA extraction from archival postmortem Bouin's fluid-fixed paraffin-embedded tissue where commercial kit yielded low success. The results obtained with the present Mini-STR PCR-multiplexes proved clearly demonstrating their usefulness in analyzing degraded DNA samples.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

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。