Use of a commercially available hydrogel as a novel DNA surface sampling tool

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.     

Characterization of human DNA in environmental samples

Environmental samples from indoor surfaces can be confounded by dust, which is composed largely of human skin cells and has been documented to contain roughly tens of micrograms of total DNA per gram of dust. This study complements previous published work by providing estimates of the quantity of amplifiable human DNA found in environmental samples from a typical indoor environment, categorized by the intensity of human traffic and visible quantity of dust. Dust was collected by surface swabbing standard 576 cm² areas in eight locations, and evaluated for total DNA quantity, presence of human DNA (mitochondrial and nuclear loci using conventional PCR), quantity of human nuclear DNA using quantitative PCR, and STR analysis. The total DNA content of 36 dust samples ranged from 9 to 28 ng/cm², and contained 0.2–1.1 pg/cm² of human DNA. Overall, human DNA was detected in 97% of 36 dust samples and 61% of samples yielded allele distributions of varying degrees of complexity when subjected to STR analysis. The implications of this study are twofold. First, the presence of dust in evidence can be a significant contamination source in forensic investigations because the human DNA component is of sufficient quality and quantity to produce allele calls in STR analysis. This can be effectively managed by implementing stringent protocols for collection and analysis of potential biological samples. A second implication is the use of dust as a source of evidence for identification of inhabitants within a defined location. In the latter case, a number of additional studies would be necessary to identify relevant pretreatments for environmental dust samples and to develop the necessary deconvolution techniques to separate the composite genotypes obtained.     

Comparison of the M-Vac® Wet-Vacuum-Based Collection Method to a Wet-Swabbing Method for DNA Recovery on Diluted Bloodstained Substrates*†‡

A wet-vacuum-based collection method with the M-Vac^® was compared to a wet-swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet-vacuum method yielded more total nuclear DNA than wet-swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet-swabbing significantly recover more DNA. The wet-vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet-vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet-vacuum method.     

The use of the M-Vac® wet-vacuum system as a method for DNA recovery

Collecting sufficient template DNA from a crime scene sample is often challenging, especially with low quantity samples such as touch DNA (tDNA). Traditional DNA collection methods such as double swabbing have limitations, in particular when used on certain substrates which can be found at crime scenes, thus a better collection method is advantageous. Here, the effectiveness of the M-Vac® Wet-Vacuum System is evaluated as a method for DNA recovery on tiles and bricks. It was found that the M-Vac® recovered 75% more DNA than double swabbing on bricks. However, double swabbing collected significantly more DNA than the M-Vac® on tiles. Additionally, it was found that cell-free DNA is lost in the filtration step of M-Vac® collection. In terms of peak height and number of true alleles detected, no significant difference was found between the DNA profiles obtained through M-Vac® collection versus double swabbing of tDNA depositions from 12 volunteers on bricks. The results demonstrate that the M-Vac® has potential for DNA collection from porous surfaces such as bricks, but that alterations to the filter apparatus would be beneficial to increase the amount of genetic material collected for subsequent DNA profiling. These results are anticipated to be a starting point to validate the M-Vac® as a DNA collection device, providing an alternative method when DNA is present on a difficult substrate, or if traditional DNA collection methods have failed.     

Assessing a novel room temperature DNA storage medium for forensic biological samples

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies.A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4× SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA.Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.     

A novel approach to obtaining reliable PCR results from luminol treated bloodstains

In recent years the forensic scientist has been afforded great advances in technology both in the detection of latent bloodstains and in acquiring reliable DNA typing results from very small pieces of physical evidence. Scientists are now able to detect minute quantities of latent bloodstains by utilizing the luminol reagent, oftentimes indicating that an attempt has been made to conceal any evidence of bloodshed. With the introduction of PCR based technology to the forensic arena, scientists are now routinely able to obtain DNA typing results from previously insufficient amounts of biological material, items as small as a single hair, saliva on a cigarette butt, or a bloodstain the size of a pin head. We present here a merging of these two advances coupled with a new collection medium for post luminol treated latent bloodstains. The forensic scientist is now able to routinely isolate and recover an adequate amount of DNA suitable for PCR typing at all of the Promega GenePrint PowerPlex 1.1 loci. In this study, several dilutions of latent bloodstains were prepared in an effort to simulate transferred bloodstains that are routinely encountered in a crime scene setting. The latent bloodstains were treated with luminol and subsequently collected using conventional cotton tipped swabs as well as a Puritan sponge tipped swab. PCR typing at the Promega GenePrint PowerPlex 1.1 loci was then attempted upon all dilutions of the latent bloodstains for both collection mediums. The results clearly indicate that it is now routinely possible to recover adequate amounts of DNA suitable for PCR typing upon post luminol treated bloodstains.     

Analysis of salivary DNA evidence from a bite mark on a body submerged in water.

A female body was recovered after approximately 5.5 h in a river with slow-moving current. On the victim's right breast, a patterned injury was discovered and determined to be from human adult teeth. Evidence was collected according to established techniques including recovery of saliva from the bite mark area despite the body being found submerged in water. DNA analysis by PCR using polymorphic STR markers revealed a DNA profile of mixed origin. In addition to the victim's DNA profile, a genotype contribution from the perpetrator was identified as a minor component. The DNA typing results from the bite mark correlated with the DNA typing results obtained from other biological trace evidence identified from the victim's genital samples. The bite mark and the DNA evidence were used to screen suspects and played an important role in obtaining resolution of this case. Consequently, it is advisable that investigators routinely swab for salivary DNA in bite mark cases, even when the amount is thought to be minimal.     

两种包装方式对小体积生物检材DNA检验的影响

目的探讨一种适用于小体积生物物证检材的包装方式。方法选择M4型螺丝钉作为研究对象,经去污消毒处理,通过手握方式转移人体脱落细胞到其表面,对螺丝钉采用两种不同的包装方式(滤纸包裹和悬空固定),经KingFisher Flex自动提取工作站进行DNA提取,从基因座等位基因检出情况、STR分型谱带峰高、均衡性等方面比较两种包装方式对螺丝钉上DNA检出率的影响。结果悬空固定包装螺丝钉实验组获得的STR分型谱带峰高、均衡性等方面均优于滤纸包裹包装实验组,其在基因座检出数量、分型完全相同样本数及有效分型样本数方面也多于滤纸包裹包装实验组。结论为降低微量生物检材DNA二次转移造成的损耗,提高小体积生物检材DNA的检出率,建议对小体积生物检材采用悬空固定方式进行物证包装。     

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.     

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.     

The Influence of Selected Fingerprint Enhancement Techniques on Forensic DNA Typing of Epithelial Cells Deposited on Porous Surfaces

Fingerprints deposited at crime scene can be a source of DNA. Previous reports on the effects of fingerprint enhancement methods have focused mainly on fingermarks deposited in blood or saliva. Here, we evaluate the effects of fingerprint enhancement methods on fingerprints deposited on porous surfaces. We performed real‐time quantification and STR typing, the results of which indicated that two methods (iodine fuming and 1,2‐indanedione in ethyl acetate enhancement) had no effect on the quantity of DNA isolated and resultant STR alleles when compared to control samples. DNA quantities and allele numbers were lower for samples enhanced with silver nitrate and 1,2‐indanedione in acetic acid when compared to control samples. Based on DNA quantity, quality, and observable stochastic effects, our data indicated that iodine fuming and 1,2‐indanedione in ethyl acetate were the preferred options for the enhancement of fingerprints on porous surfaces.     

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.     

The Correlation Between Skeletal Weathering and DNA Quality and Quantity*

Abstract: Mitochondrial DNA analysis of skeletal material is invaluable in forensic identification, although results can vary widely among remains. Previous studies have included bones of different ages, burial conditions, and even species. In the research presented, a collection of human remains that lacked major confounders such as burial age, interment style, and gross environmental conditions, while displaying a very broad range of skeletal degradation, were examined for both mitochondrial DNA (mtDNA) quality and quantity. Overall skeletal weathering, individual bone weathering, and bone variety were considered. Neither skeletal nor bone weathering influenced DNA quality or quantity, indicating that factors that degrade bone do not have the same effect on DNA. In contrast, bone variety, regardless of weathering level, was a significant element in DNA amplification success. Taken together, the results indicate that neither skeletal nor individual bone appearance are reliable indicators of subsequent mtDNA typing outcomes, while the type of bone assayed is.     

Busting the myths: DNA typeability after 48 hours of boil

The aim of our study was to monitor the quality and quantity of DNA in bone samples that were boiled for 48 h. Bos taurus bone disks were sampled every hour for 48 h. The subsequent DNA analysis used multiple mitochondrial DNA (mtDNA) targets (100–700 bp) to evaluate the quality and quantity of the DNA extracted. The DNA extracted from bone disks remained typeable after boiling for 48 h. We have proven that DNA typing results can be obtained even after long-term boiling.     

Trace DNA success rates relating to volume crime offences

In this study, 252 trace DNA samples (from handled surfaces) from 201 burglary, robbery and drugs cases were compiled to assess success rates and to interpret the value of trace DNA evidence in volume crime investigations. The average amount of DNA recovered from the trace DNA samples collected was 1.7 ng. Full or major (12 or more alleles) profiles were recovered from 14% of samples. Samples from firearms and burglary points of entry were the least successful. Mixtures were recovered from 21% of samples, presenting a case for the collection of more elimination profiles to enable more samples to be used for database purposes. The research highlighted the difficulties in collecting data relating to the success rates of samples. Computerised automation of this process would be extremely beneficial in the assistance of policy development, method application, training, and investigative usefulness.     

Effect of Sterilants on Amplification and Detection of Target DNA from Bacillus cereus Spores

To conceal criminal activity of a bioterrorist or agroterrorist, the site of pathogen generation is often treated with sterilants to kill the organisms and remove evidence. As dead organisms cannot be analyzed by culture, this study examined whether DNA from sterilant‐treated Bacillus cereus spores was viable for amplification. The spores were exposed to five common sterilants: bleach, Sterilox®, oxidizer foam (L‐Gel), a peroxyacid (Actril®), and formaldehyde vapor. The spores were inoculated on typical surfaces found in offices and laboratories to test for environmental effects. It was found that the surface influenced the efficiency of recovery of the organisms. The DNA isolated from the recovered spores was successfully detected using RT‐qPCR for all treatments except for formaldehyde, by amplifying the phosphatidylinositol phospholipase C and sphingomyelinase genes. The results demonstrated that evidence from sites treated with sterilants can still provide information on the uncultured organism, using DNA amplification.     

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.     

The utility of whole genome amplification for typing compromised forensic samples

Biological evidence has become invaluable in the crime laboratory; however, it may exist in limited quantity and/or quality. Given this, the ability to amplify total DNA obtained from evidence, in an unbiased manner, would be highly advantageous. Methods for whole genome amplification (WGA) have the potential to fulfill this role, resulting in a virtually unlimited supply of DNA. In the research presented, two WGA methods, improved primer extension preamplification and multiple displacement amplification (MDA), were tested using commercial kits. Control DNA, artificially degraded DNA, and DNA from fresh blood, aged blood, hair shafts, and aged bones underwent WGA, followed by short tandem repeat and mitochondrial DNA analysis. The methods did amplify DNA, but performed poorly on forensically relevant samples; the maximum amplicon size was reduced, and MDA often resulted in extraneous bands following polymerase chain reaction. Taken together, WGA appears to be of limited forensic utility unless the samples are of a very high quality.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

The recovery of latent fingermarks and DNA using a silicone-based casting material

There are many techniques available for the recovery of fingermarks at scenes of crime including the possibility of taking casts of the marks. Casts can be advantageous in cases where other destructive recovery techniques might not be suitable, such as when recovering finger marks deposited on valued or immobile items. In this research, Isomark (a silicone-based casting material) was used to recover casts of finger marks placed on a variety of substrates. Casts were enhanced using cyanoacrylate fuming. Good quality marks were successfully recovered from a range of smooth, non-porous surfaces. Recovery from semi-porous surfaces was shown to be inefficient. DNA was subsequently extracted from the casts using QIAamp Mini extraction kits, amplified and profiled. Full DNA profiles were obtained 34% of samples extracted.