High-resolution melt analysis for the detection of skin/sweat via DNA methylation.

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.

A data-driven, high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids.

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.

Identifying Methylation Patterns in Dental Pulp Aging: Application to Age-at-Death Estimation in Forensic Anthropology.

Age-at-death estimation constitutes one of the key parameters for identification of human remains in forensic investigations. However, for applications in forensic anthropology, many current methods are not sufficiently accurate for adult individuals, leading to chronological age estimates erring by ±10 years. Based on recent trends in aging studies, DNA methylation has great potential as a solution to this problem. However, there are only a few studies that have been published utilizing DNA methylation to determine age from human remains. The aim of the present study was to expand the range of this work by analyzing DNA methylation in dental pulp from adult individuals. Healthy erupted third molars were extracted from individuals aged 22-70. DNA from pulp was isolated and bisulfite converted. Pyrosequencing was the chosen technique to assess DNA methylation. As noted in previous studies, we found that ELOVL2 and FHL2 CpGs played a role in age estimation. In addition, three new markers were evaluated-NPTX2, KLF14, and SCGN. A set of CpGs from these five loci was used in four different multivariate regression models, providing a Mean Absolute Error (MAE) between predicted and chronological age of 1.5-2.13 years. The findings from this research can improve age estimation, increasing the accuracy of identification in forensic anthropology.

Development of a body fluid identification multiplex via DNA methylation analysis.

The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers - BCAS4, CG06379435, VE_8, and ZC3H12D - were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.

Tailoring Rheological Properties of Thermoresponsive Hydrogels through Block Copolymer Adsorption to Cellulose Nanocrystals.

This study investigates the adsorption of a block copolymer composed of a poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) cationic polyelectrolyte and a poly(di(ethylene glycol) methyl ethermethacrylate) (PDEGMA) on oxidized cellulose nanocrystals (TO-CNCs) to produce hydrogels. PDMEAMA- b-PDEGMA was synthesized by atom-transfer radical polymerization. The extent and dynamics of the adsorption of PDMAEMA- b-PDEGMA on TO-CNCs were determined by electromechanical microbalance and optical techniques. Electrostatic adsorption was identified on TO-CNCs with the quaternized block copolymer. Small-angle neutron scattering experiments were performed to investigate the polymer behavior on the TO-CNC surfaces. Depending on the temperature, block copolymer induces the aggregation of nanocrystals after adsorption by connecting CNCs bundles with block copolymer chains. A reversible liquid-to-gel transition, triggered by temperature, was clearly detected by rheological measurements for the copolymer-CNC mixtures. At the optimal copolymer to CNC ratio the viscosity increased by 4 orders of magnitude at low shear rates. These stimuli-responsive CNC-based materials could be used as injectable biomedical systems.

Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

Toward maintaining canine training aid integrity: Visualizing contamination and testing storage materials for a non-hazardous canine training aid.

Contamination of canine training aids is a pervasive issue that may lead to incorrect canine discrimination of target odors. It is therefore important to properly store training materials to maintain their integrity and efficiency. First, this study demonstrated the potential for contamination using GloGerm™ as a proxy for odor/particulate transfer. Then, eight types of containers were evaluated to determine (1) the ability to prevent odor permeation and (2) the likelihood of maintaining the ab/adsorbed odor. Lastly, a longitudinal study evaluated how the permeation of the target odor changed over time. Analysis occurred using a direct analysis in real-time mass spectrometer (DART-MS) to detect triacetone triperoxide (TATP) from the non-hazardous canine training aid known as the polymer odor capture-and-release (POCR) system. Results showed that Mylar and Opsak bags were most effective for short-term storage, maintaining low levels of ab/adsorption. Over time, the amount of TATP permeating through the primary containers and collecting in a secondary container (i.e., outer packaging) increased at 1 week and decreased thereafter (up to 4 months). The amount of TATP collecting in the primary containers, however, increased up to 1 month and decreased thereafter.

Permeation of human scent through laboratory examination gloves.

It is generally accepted in the canine detection industry that a barrier (such as a glove) should be used between a human and evidence or canine training aids in order to prevent contamination and cross-contamination as well as protect the handler from hazardous materials. However, no studies exist evaluating this assumption. Further, there is no published literature examining the different types of gloves for their utility in handling evidence or training materials used in canine detection work. This study was the first of its kind to address these gaps in the literature. First, GloGerm™ was used as a proxy for human scent and odor(s)/particulate(s) to visualize potential contamination. Then, three types of gloves (nitrile, two layers of nitrile, latex, and polyethylene) were tested for the permeation of human scent using furfural as a proof of concept, followed by pooled human sweat. Finally, the inherent odor of each glove type was identified. Two analytical techniques were used simultaneously as static and standoff dynamic detection systems, respectively: solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and direct analysis in real time-mass spectrometry (DART-MS). Using a double layer of nitrile gloves was the most effective in preventing furfural permeation from the analytical standard, while a single layer of nitrile prevented furfural from permeating from human sweat up to 2 h. Polyethylene gloves allowed the highest amount of furfural permeation but had no inherent odor detected. Headspace analysis detected two compounds for nitrile gloves and four compounds for latex gloves, but the nitrile compounds had a higher relative abundance.

Recovery of DNA from fired and unfired cartridge casings: comparison of two DNA collection methods.

For over 10 years, various studies have attempted to increase the recovery of DNA from ammunition by modifying the DNA collection, extraction, purification, and amplification procedures, with varying levels of success. This study focused on the "soaking" method of Montpetit & O'Donnell [1] and the "rinse-and-swab" method of Bille et al. [2]. First, testing for the presence of exogenous DNA, 210 boxed cartridges (brass, steel, and nickel-plated) from nine manufacturers were swabbed and DNA was extracted, concentrated, and quantified. Extracts that quantified > 0 ng/µL (44 of 210) were amplified and genotyped with GlobalFiler™. Of those, only one extract yielded two alleles indicating that the manufacturing and packaging of ammunition was virtually DNA free. Next, to obtain a baseline comparison of two DNA collection methods on a non-metallic substrate and identify a suitable number of cells to spot on cartridges, different DNA input amounts of primary human adult epidermal keratinocytes (HEKa) were tested. Thereafter, 300 brass and 300 nickel-plated, cartridges were spotted with HEKa cells containing ~5 ng of DNA, fired or unfired, and processed with either method. Finally, five methods representing hybrids of the soaking and rinse-and-swab methods were tested to determine if variations of those methods could be used to increase DNA yield and recovery. The results show that the soaking method consistently yielded more DNA than the rinse-and-swab method from a non-metallic substrate. However, the comparison study demonstrated that both methods performed comparably for cartridges. On average, the soaking method recovered 0.25 ng of DNA (5.1% recovery) and the rinse-and-swab method recovered 0.28 ng (5.8% recovery). However, average recoveries were significantly different among three analysts and considerable variation in yields were observed, possibly due to storage time. Furthermore, consistent with prior reports, the DNA recovered from brass casings was only 16% of that recovered from nickel-plated casings and the average yield of DNA from fired casings was reduced to 67% of unfired casings. Moreover, DNA extracts from brass or nickel-plated casings did not appear to contain amplification inhibitors and only 30/596 appeared severely degraded. Finally, both the published rinse-and-swab and soaking methods yielded more DNA than all modifications of the two methods. Overall, both methods yielded equivalent DNA quantities. Additionally, recovery of DNA from any given cartridge casing may be dependent on storage time as well as the skill, proficiency, and experience of the analyst and may reflect stochastic effects, particularly for casings containing low copy and/or degraded DNA.

A collaborative exercise on DNA methylation-based age prediction and body fluid typing.

DNA methylation has become one of the most useful biomarkers for age prediction and body fluid identification in the forensic field. Therefore, several assays have been developed to detect age-associated and body fluid-specific DNA methylation changes. Among the many methods developed, SNaPshot-based assays should be particularly useful in forensic laboratories, as they permit multiplex analysis and use the same capillary electrophoresis instrumentation as STR analysis. However, technical validation of any developed assays is crucial for their proper integration into routine forensic workflow. In the present collaborative exercise, two SNaPshot multiplex assays for age prediction and a SNaPshot multiplex for body fluid identification were tested in twelve laboratories. The experimental set-up of the exercise was designed to reflect the entire workflow of SNaPshot-based methylation analysis and involved four increasingly complex tasks designed to detect potential factors influencing methylation measurements. The results of body fluid identification from each laboratory provided sufficient information to determine appropriate age prediction methods in subsequent analysis. In age prediction, systematic measurement differences resulting from the type of genetic analyzer used were identified as the biggest cause of DNA methylation variation between laboratories. Also, the use of a buffer that ensures a high ratio of specific to non-specific primer binding resulted in changes in DNA methylation measurement, especially when using degenerate primers in the PCR reaction. In addition, high input volumes of bisulfite-converted DNA often caused PCR failure, presumably due to carry-over of PCR inhibitors from the bisulfite conversion reaction. The proficiency of the analysts and experimental conditions for efficient SNaPshot reactions were also important for consistent DNA methylation measurement. Several bisulfite conversion kits were used for this study, but differences resulting from the use of any specific kit were not clearly discerned. Even when different experimental settings were used in each laboratory, a positive outcome of the study was a mean absolute age prediction error amongst participant's data of only 2.7 years for semen, 5.0 years for blood and 3.8 years for saliva.