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.     

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based extraction combined with complete demineralization, that enables maximum DNA recovery and efficient elimination of PCR inhibitors. This is performed with high concentration of EDTA solution for demineralization of bone powder followed by QIAamp^® spin columns and buffers from the QIAquick^® PCR purification kit. We have successfully used this modified technique to perform STR analysis for 55-year-old skeletal remains. The results of this study will contribute to solve the forensic cases dealing with skeletal remains.     

The transfer of touch DNA from hands to glass, fabric and wood

The transfer of DNA from hands to objects by holding or touching has been examined in the past. The main purpose of this study was to examine the variation in the amount of DNA transferred from hands to glass, fabric and wood. The study involved 300 volunteers (100 for glass, 100 for fabric and 100 for wood) 50% of which were male and 50% female. The volunteers held the material for 60 s. The DNA was recovered from the objects using a minitape lift, quantified using the Quantifiler kit assay, extracted using a ‘Qiagen^® QIAamp DNA mini kit’ and amplified using the AmpFlSTR^® SGM Plus™ Amplification Kit at 28 cycles. The results show that using ANOVA there was a significant difference (F = 8.2, p < 0.05) between the three object types in the amount of DNA recovered. In terms of DNA transfer and recovery, wood gave the best yield, followed by fabric and then glass. The likelihood of success of obtaining a profile indicative of the holder was approximately 9% for glass samples, 23% for fabric and 36% for wood. There was no significant difference between the amount of DNA transferred by male or female volunteers. In this study good shedder status, as defined by obtaining useful profiles of 6 or more alleles, is estimated at approximately 22% of the population. The phenomenon of secondary transfer was observed when mixed DNA profiles were obtained but the incidence was low at approximately 10% of the total number of samples. DNA profiles corresponding to more than one person were found on objects which had been touched by only one volunteer. Although secondary transfer is possible the profiles obtained from touched objects are more likely to be as a result of primary transfer rather than a secondary source.     

A comparative study of two extraction methods routinely used for DNA recovery from simulated post coital samples

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp^® DNA mini kit (Qiagen) and Differex™ with the DNA IQ^® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH₂O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler^® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex^® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp^® DNA mini kit and involved fewer liquid transfers.     

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.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault

In cases of sexual assault involving an azoospermic assailant, vaginal swabs taken from the victim may fail to provide an autosomal DNA profile with which to search a suspect database, as the signal from any male cells present would be masked by that from the overwhelming number of female cells collected on the swab. Here, we describe a method of visually identifying diploid male cells in such samples using fluorescence in situ hybridisation, and selectively harvesting them by means of laser microdissection. This combination of techniques was tested on 26 post-coital vaginal swabs taken at a range of times after intercourse; the collected cells were then subjected to a simple lysis procedure and DNA was amplified using the AmpFlSTR^® SGMPlus^® multiplex under low copy number conditions. Useful DNA profiles were generated from samples taken up to 24 h after intercourse.     

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon^® and Microcon^® centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin^® XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin^® XS column is approximately 30 min vs. approximately 1.5 h with the Centricon^® YM-100 filter device.     

Optimization of DNA recovery from toothbrushes

In human identification, the victim's toothbrush is an invaluable personal item as the deposited cellular material contains DNA from which a reference profile can be produced. The profile obtained then allows direct comparison to be made with the profile from the unidentified body. This study was undertaken to determine the minimum number of bristle bundles that would generate a complete DNA profile. The minimum period of usage for a toothbrush to retain enough cells for genotyping was also investigated. We also tested two commonly used DNA extraction methods: QIAamp^® DNA Mini Kit and Chelex^® 100 to explore the efficiency of these protocols in recovering DNA from toothbrushes. In this experiment, volunteers brushed their teeth for 1, 7, 14, or 30 days. DNA was extracted from 5 and 10 bundles of bristles cut from the collected toothbrushes. The amount of DNA recovered was quantified by quantitative real-time PCR, and DNA genotyping was performed for each sample. Data revealed that QIAamp^® DNA Mini Kit performed better at yielding DNA in terms of purity, quantity, and quality than Chelex^® 100. It was also found that, with a suitable method of recovery, DNA samples from five bundles of bristles from all of the toothbrushes generated complete profiles. Based on the experimental results, a general guideline concerning the appropriate extraction method and the quantity of the starting material for the analysis of DNA from toothbrushes could be suggested.     

Effects of histological staining on the analysis of human DNA from archived slides

Abstract: Archived slides of cell smears treated with histological stains for sperm detection are often the only source of DNA available when cold cases are reopened. There have been conflicting reports as to the negative effects of particular histological stains on DNA recovery and quality from human cells, making stain selection an important consideration for forensic laboratories. This study investigates the effect of several staining systems on DNA recovery from histological slide samples stored from 0 to 10 weeks. DNA profiles obtained after analysis of these samples with AmpFlSTR^® Identifiler? and increased cycle AmpFlSTR^® SGM Plus? short tandem repeat (STR) profiling systems and the effects that these stains have on DNA quantity and quality over time are described. Results indicate that Christmas Tree and Hematoxylin and Eosin stains do not have significantly different effects on DNA quality after 10‐week storage of slides. This research will assist scientists to select staining systems that have minimal deleterious effects on the DNA recovered.     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

The use of Hemastix® severely reduces DNA recovery using the BioRobot® EZ1

Abstract: The choice of reagents for presumptive tests for blood, and subsequent extraction methodologies, can significantly affect both the quantity and quality of purified DNA. Blood samples directly tested with Hemastix^® yielded <1% of the DNA recovered from untested samples when purified using the Qiagen BioRobot^® EZ1 and EZ1^® DNA Investigator kit. Full short tandem repeat profiles were obtained from both tested and untested samples, suggesting that the Hemastix^® reagent(s) affect DNA binding, rather than produce DNA damage. The Hemastix^® inhibition of DNA yield could be overcome by the addition of MTL buffer to the sample prior to extraction. Laboratories may wish to modify current procedures for extracting blood samples, utilize other extraction/purification methodologies, or inform their submitting agencies to avoid direct exposure of questioned bloodstains to Hemastix^® reagents.     

Recovery of human DNA profiles from poached deer remains part 2: Improved recovery protocol without the need for LCN analysis

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis.Samples from 10 deer (40 samples total — one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5 pg ± 43.2 pg, 31 × greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpF?STR® SGM Plus® kit at 30 cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690 × 10^? 05 to 2.2706 × 10^? 14.The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.     

Validation of a 17-locus Y-STR multiplex system

Internal validation of a commercial 17-locus Y-STR system (AmpFlSTR^® Yfiler™, Applied Biosystems) has been performed on the ABI PRISM^® 3130 Genetic Analyzer for use in forensic cases. The Yfiler™ kit was validated according to SWGDAM guidelines. Our results show that it is possible to obtain full profiles with as little as 30 pg of male DNA even in the presence of 20,000-fold amounts of female DNA. Reaction volume was optimized for 10 μl. Male-male mixtures yielded full profiles of the minor contributor with 10-fold excess of the major contributor. Stutter values for each locus were determined from data generated for the population study which included Y-STR profiles from 156 caucasian males from the Montreal and Lac St.-Jean areas of Québec, Canada. The study recorded 141 different haplotypes of which 131 were unique with a haplotype diversity of 0.9965. A number of non-probative forensic samples from rape kit epithelial fractions and fingernail scrapings were also successfully tested.     

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2–6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥10²⁰) from donors that had returned non-probative LRs (<10³) by standard methods.     

Development and validation of the Cozart DDS oral fluid collection device

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart^® DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n = 134) and drug-free individuals (n = 137), using the Cozart^® DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n = 271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart^® DDS buffer and drug recovery was determined by Cozart^® enzyme immunoassays. The recovery studies showed that for amphetamine, Δ⁹THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart^® DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.     

An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler^® Duo, utilizes a TaqMan^® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY^® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

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.