Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

DNA extraction of burnt bone and teeth casework samples using bead-beating homogenization technique

Sample disruption was a necessary step for DNA isolation. Bone and teeth were useful biological sources particular in human remains and advance decomposed bodies. The compact bone and teeth required several preparation steps prior to analyzing process. However, the methods in standard protocol were laborious and time consuming. An alternating pulverization, bead beating homogenizer, was purposed in its effectiveness for forensic casework. (1) Here, we applied this technique to the burnt cracked bone and tooth that recovered from house fire for forensic DNA analysis. After cleansed an external surface, the eight multidirectional motion tissue homogenizer, Precellys® evolution, was utilized to pulverize bone and tooth followed by a DNA extraction and amplification. For detection with a capillary electrophoresis, full profiles of autosomal STRs and Y-chromosomal STRs were recovered from tooth sample but the partial profile STR was demonstrated in bone sample. The new technique in bone homogenization was less time consuming (around 30 s), less exposure to chemical agents (no need of liquid nitrogen), high efficiency, with high-throughput productivity.     

An STR Melt Curve Genotyping Assay for Forensic Analysis Employing an Intercalating Dye Probe FRET*

Abstract: The most common markers used in forensic genetics are short tandem repeats (STRs), the alleles of which are separated and analyzed by length using capillary electrophoresis (CE). In this work, proof of concept of a unique STR genotyping approach has been demonstrated using asymmetric PCR and a fluorescence resonance energy transfer (FRET)‐based hybridization analysis that combines fluorophore‐labeled allele‐specific probes and a DNA intercalating dye (dpFRET) in a melt match/mismatch analysis format. The system was successfully tested against both a simple (TPOX) and a complex (D3S1358) loci, demonstrated a preliminary detection limit of <10 genomic equivalents with no allelic dropout and mixture identification in both laboratory‐generated and clinical samples. With additional development, this approach has the potential to contribute to advancing the use of STR loci for forensic applications and related fields.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes

A single nucleotide polymorphism (SNP) multiplex has been developed to analyse highly degraded and low copy number (LCN) DNA template, i.e. <100 pg, for scenarios including mass disaster identification. The multiplex consists of 20 autosomal non-coding loci plus Amelogenin for sex determination, amplified in a single tube PCR reaction and visualised on the Applied Biosystems 3100 capillary electrophoresis (CE) system. Allele-specific primers tailed with shared universal tag sequences were designed to speed multiplex design and balance the amplification efficiencies of all loci through the use of a single reverse and two differentially labelled allele denoting forward universal primers. As the multiplex is intended for use with samples too degraded for conventional profiling, a computer program was specifically developed to aid interpretation. Critical factors taken into account by the software include empirically determined extremes of heterozygous imbalance (Hb) and the drop-out threshold (Ht) defined as the maximum peak height of a surviving heterozygous allele, where its partner may have dropped out. The discrimination power of the system is estimated at 1 in 4.5 million, using a White Caucasian population database. Comparisons using artificially degraded samples profiled with both the SNP multiplex and AMPFISTR SGM plus (Applied Biosystems) demonstrated a greater likelihood of obtaining a profile using SNPs for certain sample types. Saliva stains degraded for 147 days generated an 81% complete SNP profile whilst short tandem repeats (STRs) were only 18% complete; similarly blood degraded for 243 days produced full SNP profiles but only 9% with STRs. Reproducibility studies showed concordance between SNP profiles for different sample types, such as blood, saliva, semen and hairs, for the same individual, both within and between different DNA extracts.     

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security

Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO^® 150. This modified application of the PrepFiler™ technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch^® Technology (CST) from Invitrogen.     

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.     

Effectiveness of Single‐Nucleotide Polymorphisms to Investigate Cattle Rustling

Short tandem repeats (STR)s have been the eligible markers for forensic animal genetics, despite single‐nucleotide polymorphisms (SNP)s became acceptable. The technology, the type, and amount of markers could limit the investigation in degraded forensic samples. The performance of a 32‐SNP panel genotyped through OpenArrays^TM (real‐time PCR based) was evaluated to resolve cattle‐specific forensic cases. DNA from different biological sources was used, including samples from an alleged instance of cattle rustling. SNPs and STRs performance and repeatability were compared. SNP call rate was variable among sample type (average = 80.18%), while forensic samples showed the lowest value (70.94%). The repeatability obtained (98.7%) supports the used technology. SNPs had better call rates than STRs in 12 of 20 casework samples, while forensic index values were similar for both panels. In conclusion, the 32‐SNPs used are as informative as the standard bovine STR battery and hence are suitable to resolve cattle rustling investigations.     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

DNA IQ^TM SYSTEM在疑难指甲DNA检验中的应用

目的探索DNA IQTMSYSTEM在疑难指甲DNA提取中的应用。方法 15份疑难指甲采用Chelex方法检验没有成功获得STR分型图谱,采用DNA IQTMSYSTEM提取法并纯化,采用Identifiler PLUS试剂盒进行复合扩增,产物经ABI3130XL型DNA基因分析仪检测。结果成功获得15例疑难指甲的STR基因座DNA分型。结论 DNA IQTMSYSTEM方法能快速、有效提取疑难指甲DNA进行STR分型。     

Validation of STR typing by capillary electrophoresis

With the use of capillary electrophoresis (CE), high-resolution electrophoretic separation of short tandem repeat (STR) loci can be achieved in a semiautomated fashion. Laser-induced detection of fluorescently labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by capillary electrophoresis were investigated using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). An internal size standard was used with each run to effectively normalize mobility differences among injections. Alleles were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demonstrate that allele sizes were reproducible, with standard deviations typically less than 0.12 bases for fragments up to 317 bases in length (largest allele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all alleles that are the same length should fall within the measurement error window of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were also accurately typed by the analytical software. Alleles differing in size by one base could be resolved in two-donor DNA mixtures in which the minor component comprised > or = 5% of the total DNA. Furthermore, the quantitative data format (i.e., peak amplitude) can in some instances assist in determining individual STR profiles in mixed samples. DNA samples from previously typed cases (typed for RFLP, AmpliType PM+DQA1, and/or D1S80) were amplified using AmpFlSTR Profiler Plus and COfiler and were evaluated using the ABI Prism 310. Most samples yielded typable results. Compared with previously determined results for other loci, there were no discrepancies as to the inclusion or exclusion of suspects or victims. CE thus provides efficient separation, resolution, sensitivity and precision, and the analytical software provides reliable genotyping of STR loci. The analytical conditions described are suitable for typing samples such as reference and evidentiary samples from forensic casework.     

Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt.

The short tandem repeat loci (STRs) D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and a locus allowing for sex-discrimination (amelogenin) can be co-amplified by the polymerase chain reaction using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA) and subsequently typed using capillary electrophoresis (ABI Prism 310 Genetic analyzer, Perkin Elmer Applied Biosystems, San Jose CA). To establish databases for these loci for an Arab population sample from Egypt, 140 unrelated persons were typed. Analysis of these data revealed that all loci except for VWA were in Hardy-Weinberg equilibrium, that the combined mean paternity exclusion chance (MEC) was 0.999875 and that the combined discriminating power (DP) was 2.635 x 10(-11). The allelic distributions found in the Egyptian sample were significantly different at four loci from those found for an Austrian Caucasian population, at all nine loci from an African-American sample and at six of six loci from a Chinese sample. No evidence of linkage equilibrium between any of the co-amplified loci was found. Our results support that the combination of multiplex PCR and capillary electrophoresis can both save time and yield excellent results for paternity testing and stain analysis.     

Identification of Acer rubrum using amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) analysis of botanical forensic evidence provides a means of obtaining a reproducible DNA profile in a relatively short period of time in species for which no sequence information is available. AFLP profiles were obtained for 40 Acer rubrum trees. Leaf material from five additional species was also typed. Genomic DNA was isolated using the DNeasy Plant Miniprep Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (pre-amplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. A number of Acer rubrum species-specific peaks were identified. In addition, within this closed set of samples, 15 of 16 (93.8%) blind samples were correctly identified. AFLP data can be used to determine the species of botanical evidence or to associate a sample to a source. This information can be used in forensic investigations to link a piece of evidence with a particular location or suspect.     

Internal validation of reduced PCR reaction volume of the Qiagen Investigator® Argus X-12 QS Kit from blood samples on FTA cards

The onus of proof in criminal cases is beyond any reasonable doubt, and the issue on the lack of complete internal validation data can be manipulated when it comes to justifying the validity and reliability of the X-chromosomal short tandem repeats analysis for court representation. Therefore, this research evaluated the efficiency of the optimized 60% reduced volumes for polymerase chain reaction (PCR) amplification using the Qiagen Investigator® Argus X-12 QS Kit, as well as the capillary electrophoresis (CE) sample preparation for blood samples on Flinder's Technology Associates (FTA) cards. Good-quality DNA profile (3000–12,000 RFU) from the purified blood sample on FTA card (1.2 mm) were obtained using the optimized PCR (10.0 μL of PCR reaction volume and 21 cycles) and CE (9.0 μL Hi-Di™ Formamide and 0.3 μL DNA Size Standard 550 [BTO] and 27 s injection time) conditions. The analytical and stochastic thresholds were 100 and 200 RFU, respectively. Hence, the internal validation data supported the use of the optimized 60% reduced PCR amplification reaction volume of the Qiagen Investigator® Argus X-12 QS Kit as well as the CE sample preparation for producing reliable DNA profiles that comply with the quality assurance standards for forensic DNA testing laboratories, while optimizing the analytical cost.     

Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep kit

Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate amplicons by length and fluorescent labeling. In recent years new methods, including massively parallel sequencing (MPS), have been developed which increased the discriminative power of STRs through sequencing. MPS also offers the opportunity to test more genetic markers in a run than is possible with standard CE technology. Verogen’s ForenSeq™ DNA Signature Prep kit includes over 150 genetic markers [STRs and single nucleotide polymorphisms (SNPs)]. Further, MPS separation depends on sequences rather than lengths; therefore, amplicons can be small or even of the same lengths. These improvements are advantageous when testing challenging forensic samples that could be severely degraded.This study tested the ForenSeq™ DNA Signature Prep kit in repeated experimental runs on series of degraded DNA samples, ranging from mild to severe degradation, as well as 24 mock case-type samples, derived from bones, blood cards, and teeth. Despite passing the quality metrics, positive controls (2800 M) showed drop-outs at some loci, mostly SNPs. Sequencing DNA samples repeatedly in two experimental runs as well as sequencing one pooled library in triplicate led to the assumption that spurious alleles of the Y-STRs in this study were not a result of sequencing artifacts but could be due to sequence structures (e.g. duplications, palindromes) of the Y-chromosome and/or might be accumulated during library preparation.Two sets of serially degraded DNA samples revealed that dropped-out loci were primarily loci with long amplicons as well as low read numbers (coverage), e.g. PentaE, DXS8378, and rs1736442. STRs started to drop out at degradation indices (DIs) > 4. However, severely degraded DNA (DI: 44) still resulted in 90% of the 20 CODIS loci, while only 35% were obtained using Promega’s PowerPlex® Fusion kit, a current standard CE kit. Mock case-type samples confirmed these results. ForenSeq™ DNA Signature Prep kit demonstrated that it can be successfully used on degraded DNA samples. This study may be helpful for other laboratories assessing and validating MPS technologies.     

High‐Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science

Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high‐resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual‐dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics.     

Grading a rape case followed by death from the study of autosomal STRs and STRs of the Y chromosome—Case study

Two women were found dead inside a residence. Choke causes death in one that had been naked in a bed and contusion injury in another that was found on a sofa. Were received samples of vaginal and anal swabs of the two victims of homicide with suspected of having suffered sexual violence. References also received samples of two victims and a suspect. We performed genetic analysis for identification of samples from the meeting of any possibility of overlap between patterns and profiles of sequences of deoxyribonucleic acid (DNA) based on genetic relationship between those involved. The reference samples were subjected to the procedure of extraction of nuclear DNA by Chelex method and the swabs samples by differential extraction. For all the samples were performed for amplification of STRs loci and autosomal STRs of chromosome Y. The profiles of DNA sequences were obtained by the Polymerase Chain Reaction (PCR), using sequences starting with marked substances emitting fluorescence detected by reading the optical laser in 3100 Avant automatic sequencer from Applied Biosystems. The information of consecutive loci of Short Repeats or STRs of autosomal chromosomes and the Y chromosome was obtained using the systems or products sold in multilocus, methodologies recommended by the supplier and valid for analysis of DNA. We used the multilocus Identifiler and YFiler system of Applied Biosystems to the amplification of samples. The validation of results has shown a genetic profile in male anal secretion of the victims with a complete coincidence with the suspect.     

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。     

Population data for MiniNC01 in a population sample from North-eastern Italy and their use in neoplastic tissues fixed in formalin and embedded in paraffin

To establish a database for the three MiniNC01 loci D10S1248, D14S1434, D22S1045 in a population sample from North-eastern Italy, 102 unrelated individuals were typed. DNA was amplified in a multiplex reaction with subsequent automatic detection using capillary electrophoresis. The obtained data give a contribution to the definition of Italian population miniSTRs allele frequencies for the three analysed loci. These three MiniSTRs were tested on 21 neoplastic tissues and the obtained genotypes were compared to those obtained from normal tissue. Only 3 cases (14.28%) gave a different genotype suggesting a better performance of these markers than traditional STRs.     

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler^® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR^® Identifiler^® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.