An investigation of two methods of DNA recovery from fired and unfired 9 mm ammunition

Cartridge cases are often recovered from crime scenes involving firearms and, in the United Kingdom (where gun possession is strictly controlled), these are commonly from 9 mm calibre ammunition. The ability to obtain informative DNA profiles from touch DNA on recovered cartridges could have a significant impact on the investigation of that type of offence. However, this avenue may not be routinely considered as investigators in the UK have historically had a low expectation of obtaining useful DNA profiles. This stance may not be unreasonable given that (a) only trace amounts of DNA are likely to have been transferred onto the cartridge cases through handling; and (b) when the cartridge is spent, the potential deterioration of that DNA caused by the act of discharging the weapon.We introduce a novel semi-automatable method using direct lysis for the recovery of DNA from ammunition and compare it with a traditional double-swabbing method (using wet and dry swabs). DNA profiling of the DNA recovered using both methods was carried out using the ESI17 FAST STR system (Promega). This demonstrated a significant increase in DNA recovery using the direct lysis approach, and correspondingly improved STR results.We also investigated the effect on the recovery and profiling of DNA from fired, and unfired, 9 mm cartridges using the direct lysis technique. These results demonstrate that DNA suitable for STR analysis can still be recovered from fired ammunition with only slightly reduced yields compared to unfired ammunition. In these experiments, the handler of the ammunition was most commonly either the sole contributor or the major contributor to the recovered DNA profile.     

Quantitative PCR overestimation of DNA in samples contaminated with tin

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an “inhibition study” and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.     

A Comparison of Fingerprint Sweat Corrosion of Different Alloys of Brass

Fingerprint sweat from 40 donors was deposited onto samples of five α and α + β phase brasses, comprising five alloys with different copper and zinc concentrations, two of which also had the addition of small concentrations of lead. Visual grading of the visibility of the corrosion revealed that brasses with the least amount of zinc produced the most visible and fully formed fingerprints from the most donors. Consideration of previously reported mechanisms for the corrosion of brass suggests red copper (I) oxide as a likely corrosion product for low zinc brasses, and a consideration of the color, composition, and solubility of fingerprint sweat corrosion products suggests that copper (I) oxide produces good contrast and visibility with the brass substrate. Scanning electron microscope images of the corrosion of all five alloys confirmed the enhanced contrast between corroded and uncorroded areas for low zinc alloys.     

1,2,4‐Triazine‐Based Chromogenic Reagents for the Detection of Microtraces of Various Metals Left on Human Skin*

Abstract: This article extends the application of 1,2,4‐triazine‐based chromogenic reagents to the detection of nonferrous metal traces left on contact with canvas and human skin. The possibility of detection of iron traces resulting from contact with objects made of stainless steel was investigated as well. Additionally, the ability of triazines to form chromatic complexes with Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Cr³⁺, and Al³⁺ ions was studied spectrophotometrically. Molar absorption coefficients, ranging from 8.8 to 29.9 × 10³/M/cm, provide high sensitivity of 1,2,4‐triazines toward nonferrous ions, thus, enabling the detection at concentrations as low as a few μM. The method was sensitive enough to detect traces resulting from a 1‐min contact with a stainless steel made object, which is commonly considered as a corrosion‐resistant material. The amounts of metal ions transferred to the skin after a 2‐min contact with objects made of brass, zinc, and copper were sufficient to develop chromatic imprints.     

Effect of chloride ion concentration on the galvanic corrosion of α phase brass by eccrine sweat

Inductively coupled plasma mass spectrometry measurement of the relative concentration of sodium, chloride, calcium, and potassium ions in eccrine sweat deposits from 40 donors revealed positive correlations between chloride and sodium (ρ = 0.684, p < 0.01) and chloride and calcium ions (ρ = 0.91, p < 0.01). Correlations between ion concentration and the corrosion of α phase brass by the donated sweat were investigated by visual grading of the degree of corrosion, by measuring the copper/zinc ratio using energy-dispersive X-ray spectroscopy, and from a measurement of the potential difference between corroded and uncorroded brass when a large potential was applied to the uncorroded brass. An increasing copper/zinc ratio (indicative of dezincification) was found to correlate positively to both chloride ion concentration and visual grading of corrosion, while visual grading gave correlations with potential difference measurements that were indicative of the preferential surface oxidation of zinc rather than copper.     

Comparison of three DNA extraction methods for three different types of fired and unfired ammunition

When handling ammunition for gun loading, epithelial cells from the hands can become adhered to the metal surface, and this trace is a potential source of DNA. This work aimed to compare the efficiency of three DNA extraction methods from fired cartridge cases from three different types of firearms: a 12-gauge shotgun, a point 40 S&W pistol, and a 7.62 mm rifle. Nine volunteers were involved in this study handling 42 pieces of ammunition overall. The unfired ammunition was handled by a known good donor, and we used this data for comparison. DNA profiling was carried out with EZ1 DNA Investigator Kit for EZ1 Advanced XL automated DNA extraction, QIAmp DNA Investigator kit for a non-automated silica-based membrane column method, and direct lysis protocol for a non-automated in-house one. Samples were collected with 0.5 × 0.5 cm pieces of FTA filter paper moistened with distilled water. Quantiplex Pro RGQ kit and Fusion Powerplex 6C were used for genotyping samples. QIAmp DNA Investigator method resulted in the best number of alleles recovered for both conditions tested, both unfired and fired ammunitions: 77 % vs. 19.3 %, followed by the automated extraction (28.6 % vs. 4.3 %) and lysis protocol (0 % vs. 3.9 %). Degradation data from fired cartridge cases were 27 % for column method, 50 % for lysis protocol, and 87 % for EZ1 kit. Kruskal-Wallis test for mean DNA concentration from these samples returned p < 0.05, and Dunn’s multiple comparison test indicated a significant difference between calibers 0.40 S&W and 12-gauge shotgun from lyses protocol method. We did not detect any other significant differences on the test. The 12-gauge shotgun cartridge cases resulted in a high number of alleles overall (56.8 %). The numerous steps for DNA extraction and purification in the column method may explain its better performance. Although the results obtained indicate that all methods be used for DNA extraction from this type of evidence, the silica-based membrane column method appears to be more efficient.     

Effect of Temperature on the Visualization by Digital Color Mapping of Latent Fingerprint Deposits on Metal

Visualization of fingerprint deposits by digital color mapping of light reflected from the surface of heated brass, copper, aluminum, and tin has been investigated using Adobe^® Photoshop^®. Metals were heated to a range of temperatures (T) between 50°C and 500°C in 50°C intervals with enhancement being optimal when the metals are heated to 250°C, 350°C, 50°C, and 300°C, respectively, and the hue values adjusted to 247°, 245°, 5°, and 34°, respectively. Fingerprint visualization after color mapping was not degraded by subsequent washing of the metals and color mapping did not compromise the visibility of the fingerprint for all values of T. The optimum value of T for fingerprint visibility is significantly dependent of the standard reduction potential of the metal with Kendall's Tau (τ) = 0.953 (p < 0.001). For brass, this correlation is obtained when considering the standard reduction potential of zinc rather than copper.     

Combined DNA Typing and Protein Identification from Unfired Brass Cartridges,,,

Biological evidence analysis from contact traces is adversely affected by low quantity and quality of DNA. Proteins in these samples contain potentially individualizing information and may be particularly important for difficult surfaces such as brass, where DNA may yield incomplete profiles. In this study, touched unfired brass cartridges were sampled using dry tape or wet swabs and analyzed by separating DNA and protein from the same collected material, thus producing both genomic and proteomic information. DNA recovery was similar for both collection methods, with tape yielding an average of 1.36 ± 1.87 ng and swabs, 1.34 ± 3.04 ng. Analysis by mass spectrometry identified 95 proteins, with the two collection methods showing no significant difference (p = 0.76) in the average number of collected proteins: 44.5 ± 10.9, (tape) versus 47.9 ± 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.     

Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2 h and 30 min with ≤0.8 bp allele typing accuracy. The minimal amount of DNA required for a complete DNA profile is 100 copies. To critically evaluate the capabilities of our portable microsystem as well as its compatibility with crime scene investigation processes, real-time STR analyses were carried out at a mock crime scene prepared by the Palm Beach County Sheriff's Office (PBSO). Blood stain sample collection, DNA extraction, and STR analyses on the portable microsystem were conducted in the field, and a successful “mock” CODIS hit was generated on the suspect's sample within 6 h. This demonstration of on-site STR analysis establishes the feasibility of real-time DNA typing to identify the contributor of probative biological evidence at a crime scene and for real-time human identification.     

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.     

PE‐Swab Direct STR Amplification of Forensic Touch DNA Samples

The PE‐Swab direct STR amplification workflow was developed to process low‐level “touch DNA” samples. In this workflow, a forensic sample is first collected on a 4‐mm PE‐Swab (a novel sample collection device); two 2‐mm punches containing collected samples are then generated from the PE‐Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE‐Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE‐Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE‐swab workflow is 3 times higher than that from the conventional workflow with both low‐level single source and two‐contributor mixture samples tested in this study.     

Direct PCR Improves the Recovery of DNA from Various Substrates

This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs^™. Swabs (n = 90) were either processed using the DNA IQ^™ kit or, for direct PCR, swab fibers (~2 mm²) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.     

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.     

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR^® Identifiler^® Plus for 28 cycles. Following amplification, samples were denatured at 99.9 °C for 5 min and incubated with one unit of DSN at 62 °C in a 28 μl volume for 1 min. Nuclease activity was terminated through the addition of equal volume of 10 mM EDTA and 95 °C incubation for 2 min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.     

A Preliminary Study on Vacuum Metal Deposition as a Standalone Method for Enhancement of Fingermarks on Ballistic Brass Materials

In order to assess the efficacy of vacuum metal deposition (VMD) as a technique to develop fingermarks on ballistic metallic surfaces, a preliminary study using six donors (three male & three female) was conducted. Using a sequential metal deposition process, two metal combinations were studied—gold/zinc and silver/zinc. Results indicate the potential of this technique, by developing identifiable fingermarks on brass metal disks aged from a few days up to more than a month old. As the development of fingermarks on fired (brass) cartridge cases is an area of interest, a further study was conducted where a total of 20 fingermarks were deposited on cases. After firing, second level fingermark characteristics were successfully observed on cartridge cases highlighting its potential as a fingermark enhancement method for ballistic brass materials. Further work is required to fully evaluate the VMD process and its reliability as a fingermark enhancing method on ballistic surfaces.     

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.     

Comparison of operational DNA recovery methods: Swabs versus tapelifts

It is routine among many jurisdictions to recover DNA using tapelifts on porous substrates (e.g. clothing) and swabs on non-porous substrates (e.g. tool handles). Here, we examine this by comparing the efficiency of the NSW jurisdiction’s specific swabbing and tapelift techniques on a range of porous and non-porous substrates. To test DNA recovery efficiency, 30 μl aliquots of 1:50 and 1:100 saliva dilutions were deposited onto the substrates, left to dry overnight, recovered, extracted, quantified and a subset profiled. Tapelifts recovered more DNA and DNA profiles with more detectable alleles than swabs for both saliva dilutions on porous substrates. For non-porous substrates, similar DNA quantities and profiles were generally recovered with both methods for both saliva dilutions. These data underpin current practices to recover DNA using tapelifts for porous substrates and swabs for non-porous substrates. These data also revealed severe degradation of DNA recovered from brass, supporting the on-going need to improve DNA recovery and analysis methods for brass substrates.     

Interrogation of short tandem repeats using fluorescent probes and melting curve analysis: A step towards rapid DNA identity screening

Current forensic DNA profiling methods rely on the analysis of samples at specialised laboratories with an average turnaround time of several days. The ability to rapidly determine a partial profile of short tandem repeats at the point-of-arrest would be of great benefit to police forces around the world, for example enabling a suspect to be rapidly included or excluded from an investigation. We have developed a homogeneous PCR method for the interrogation of STR loci utilising fluorescent oligonucleotide probes and melting curve analysis. Alleles of the D18S51, TH01 and D8S1179 loci were differentiated and identified on the basis of target length and probe melting temperature. Assay performance was evaluated by comparing melting peak data with the AmpFlSTR^® SGM Plus^® system. The method is compatible with direct analysis of unpurified buccal swab samples, enabling a partial STR profile to be generated within 1 h.     

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.     

The Successful Recovery of Low Copy Number and Degraded DNA from Bones Exposed to Seawater Suitable for Generating a DNA STR Profile

A universal method allowing for DNA profiling from bones exposed to seawater has not been reported yet. This study refers on the identification of a body immersed in seawater for 8 months. The biological material for identification was the mandibular body, usually characterized by low success rates of DNA analysis. Initially, two extraction protocols were performed with negative results: one used for bones immersed in fresh water and a silica‐column procedure. A third protocol was performed, which combined the extraction of a higher amount of bone powder, the use of multi‐silica‐based extraction columns followed by a concentration step. This protocol allowed to obtain low copy number DNA and to generate a 12‐loci STR profile by combining conventional STR typing and mini‐STR technologies. This protocol could be suitable when human bones have been exposed to severe environmental conditions, and the available nuclear DNA is highly degraded and in low copy number.