Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Recording and analysing DNA from osteocytes in resin-embedded bone samples.

We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI (4',6-diamidino-2-phenylindole). Knowledge of the presence and number of osteocytes is key to any success in subsequent DNA profiling. Osteocytes are most numerous cells and thus the main source of DNA in bone samples, which can be preserved for histological analyses. Archived samples are either fixed in formalin or preserved in ethanol prior to embedding in resin. These resin-embedded samples are potentially used as ante mortem reference samples. Cases of a missing person investigation are one example where this type of preserved reference material may be of value. When resin is required for sample preservation it represents a problem for subsequent DNA profiling, if needed as a reference sample in human identification. It is essential therefore to remove the resin prior to DNA analyses as resin is a known inhibitor of DNA profiling. Current methods of resin removal are lengthy and require toxic chemicals. This report describes a simplified process to remove resin and visualise the location of nucleated osteocytes. Eight sections of bone samples at 5-µm thickness were stained with DD and DAPI. A further three samples were processed using a formalin-fixed method and three additional samples treated following an ethanol-preserved method (11 samples for both the formalin-fixed and 11 for the ethanol-preserved with eight in common). The location and number of nuclei could be recorded clearly due to the fluorescence created by the dye binding to DNA. The number of stained nuclei correlated with the mass of DNA isolated from the sections (r = 0.873, p = 1.21 × 10-10). A significant difference between the degradation indices of two groups (p = 8.505 × 10-5) showed that ethanol preservation is a preferred method to yield DNA of the quality needed for subsequent short tandem repeats (STR) profiling. Ten of the 11 samples isolated using the ethanol-preserved process recorded a complete STR profile (30/30 alleles), whereas eight of the formalin-fixed samples generated full profiles, and only one of the 11 samples amplified less than 23 alleles. Both the ethanol-preserved and formalin-fixed methods are an improvement on current methods by removing the need for strong solutes in resin removal, and the method leads to STR profiles from resin-embedded bone samples within 24 h.

Y-SNP Haplogroup Hierarchy Finder: a web tool for Y-SNP haplogroup assignment.

The application of massively parallel sequencing (MPS) data from whole genomes has allowed very many more Y-SNP loci to be genotyped simultaneously than previously possible. Although this greatly increases the resolution of Y-SNP haplogroups to link common ancestors, it remains a great challenge to provide a phylogenetic tree to clearly display the relationship of varying haplogroups. Y-SNP Haplogroup Hierarchy Finder is a web tool to generate hierarchical haplogroups based on Y-SNP data with the derived allele at the terminal of a haplogroup tree. The input data can include that from whole-genome sequencing. Confidence in assignment using Y-SNP Haplogroup Hierarchy Finder was demonstrated using Y-SNP genotypes of 1233 samples, sourced from the 1000 genomes project phase 3, used to generate the expected haplogroups. The outcome includes 2 reports: a 'Haplogroup Report' lists mutation types from the submitted Y-SNPs and their corresponding haplogroups, and a 'Haplogroup Hierarchy Report' lists all possible hierarchical haplogroups and ranks the three most supported haplogroups. Each layer of the descending haplogroups from one step to the next is shown and the supporting numbers of Y-SNPs are also included in these reports. All haplogroups that exhibited a clear relationship between the ancestral through to the derived SNPs can be clustered into a hierarchy of haplogroups. The assigned 1233 haplogroups were compared with 2 other software programs designed to assemble haplogroups, which resulted in one where there were many differences and the other one where there was only minor difference. The advantage of this web-based tool is that it provides an easy way to assign Y-SNP haplogroup based on the visualized hierarchical pattern.

A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa.

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Development of an STR panel for a non-native population of an endangered species.

BACKGROUND: The establishment of non-native populations of threatened and legally protected species can have many implications for the areas where these species have been introduced. Non-native populations of threatened species have the potential to be exploited and therefore the subject of legal protection, while conversely, if they have become invasive in their introduced range, there is the likelihood that population control will be carried out to reduce abundance and negative impacts associated with introduced species. From both a legal and invasive species monitoring standpoint, it is important to know how many individuals are present. METHODS AND RESULTS: Short tandem repeats (STRs) were developed for the hog deer, an endangered species that was introduced following European settlement to Victoria, Australia using Illumina MiSeq sequencing technology. These markers were combined with previous STRs characterised for hog deer to create a 29-plex identification system. A total of 224 samples were genotyped across the population in Victoria, and further analyses of null allele frequencies, deviation from Hardy-Weinberg equilibrium, and the removal of monomorphic or low amplifying markers resulted in a final marker panel of 15 loci. Despite low values for number of alleles at each locus (2-4), probability of identity showed sufficient discrimination power, with an average probability of identity at 2.94 × 10-6, and a probability of sibling identity of 8.9 × 10-4 across all sites. CONCLUSIONS: It is feasible to create an informative DNA profiling system that can distinguish between individuals for applications in both wildlife forensic and population control research.

Discrimination of highly degraded, aged Asian and African elephant ivory using denaturing gradient gel electrophoresis (DGGE).

BACKGROUND: Elephant populations have greatly reduced mainly due to illegal poaching for their ivory. The trade in elephant products is protected by national laws and CITES agreements to prevent them from further decline. For instance, in Thailand, it is illegal to trade ivory from African elephants; however, the law allows possession of ivory from Asian elephants if permission has been obtained from the authorities. As such, means of enforcement of legislation are needed to classify the legal status of seized ivory products. Many DNA-based techniques have been previously reported for this purpose, although all have a limit of detection not suitable for extremely degraded samples. AIM: We report an assay based on nested PCR followed by DGGE to confirm the legal or illegal status of seized ivory samples where it is assumed that the DNA will be highly degraded. METHOD AND RESULTS: The assay was tested on aged ivory from which the assay was tested for reproducibility, specificity, and, importantly, sensitivity. Blind testing showed 100% identification accuracy. Correct assignment in all 304 samples tested was achieved including confirmation of the legal status of 227 highly degraded, aged ivories, thus underlining the high sensitivity of the assay. CONCLUSION AND RECOMMENDATION: The research output will be beneficial to analyze ivory casework samples in wildlife forensic laboratories.

The development of a tool to predict temperature-exposure of incinerated teeth using colourimetric and hydroxyapatite crystal size data.

This study presents a novel tool to predict temperature-exposure of incinerated pig teeth as a proxy for understanding impacts of fire on human teeth. Previous studies on the estimation of temperature-exposure of skeletal elements have been limited to that of heat-exposed bone. This predictive tool was developed using a multinomial regression model of colourimetric and hydroxyapatite crystal size variables using data obtained from unheated pig teeth and teeth incinerated at 300 °C, 600 °C, 800 °C and 1000 °C. An additional variable based on the observed appearance of the tooth was included in the tool. This enables the tooth to be classified as definitely burnt (600 °C-1000 °C) or uncertain (27 °C/300 °C). As a result, the model predicting the temperature-exposure of the incinerated teeth had an accuracy of 95%. This tool is a holistic, robust and reliable approach to estimate temperature of heat-exposed pig teeth, with high accuracy, and may act as a valuable proxy to estimate heat exposure for human teeth in forensic casework.

Population inference based on mitochondrial DNA control region data by the nearest neighbors algorithm.

Population and geographic assignment are frequently undertaken using DNA sequences on the mitochondrial genome. Assignment to broad continental populations is common, although finer resolution to subpopulations can be less accurate due to shared genetic ancestry at a local level and members of different ancestral subpopulations cohabiting the same geographic area. This study reports on the accuracy of population and subpopulation assignment by using the sequence data obtained from the 3070 mitochondrial genomes and applying the K-nearest neighbors (KNN) algorithm. These data also included training samples used for continental and population assignment comprised of 1105 Europeans (including Austria, France, Germany, Spain, and England and Caucasian countries), 374 Africans (including North and East Africa and non-specific area (Pan-Africa)), and 1591 Asians (including Japan, Philippines, and Taiwan). Subpopulations included in this study were 1153 mitochondrial DNA (mtDNA) control region sequences from 12 subpopulations in Taiwan (including Han, Hakka, Ami, Atayal, Bunun, Paiwan, Puyuma, Rukai, Saisiyat, Tsou, Tao, and Pingpu). Additionally, control region sequence data from a further 50 samples, obtained from the Sigma Company, were included after they were amplified and sequenced. These additional 50 samples acted as the "testing samples" to verify the accuracy of the population. In this study, based on genetic distances as genetic metric, we used the KNN algorithm and the K-weighted-nearest neighbors (KWNN) algorithm weighted by genetic distance to classify individuals into continental populations, and subpopulations within the same continent. Accuracy results of ethnic inferences at the level of continental populations and of subpopulations among KNN and KWNN algorithms were obtained. The training sample set achieved an overall accuracy of 99 to 82% for assignment to their continental populations with K values from 1 to 101. Population assignment for subpopulations with K assignments from 1 to 5 reached an accuracy of 77 to 54%. Four out of 12 Taiwanese populations returned an accuracy of assignment of over 60%, Ami (66%), Atayal (67%), Saisiyat (66%), and Tao (80%). For the testing sample set, results of ethnic prediction for continental populations with recommended K values as 5, 10, and 35, based on results of the training sample set, achieved overall an accuracy of 100 to 94%. This study provided an accurate method in population assignment for not only continental populations but also subpopulations, which can be useful in forensic and anthropological studies.

A novel approach for rapid cell assessment to estimate DNA recovery from human bone tissue.

We report on the use of a DNA staining dye to locate and record nucleated osteocytes and other bone-related cells within sections of archived formalin-fixed and paraffin-embedded human tibia from which informative DNA profiles were obtained. Eleven of these archived tibia samples were sectioned at a thickness of 5 µm. Diamond™ Nucleic Acid Dye was applied to the sections and cells within the matrix of the bone fluoresced so that their location and number of cells could be photographed. DNA was isolated from these 11 samples using a standard extraction process and the yields were quantified by real-time PCR. Complete STR profiles were generated from ten bone extracts where low-level inhibition was recorded with an incomplete STR profile obtained from one sample with higher inhibition. The stained image of this sample showed that few cells were present. There was a significant relationship between the number of DD-stained cells and the number of alleles obtained (p < 0.05). Staining cells to determine the prevalence of bone cell nuclei allows a triage of samples prior to any subsequent DNA profiling.

Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults.

Identification of semen and then spermatozoa is essential to verify that sexual activity has occurred in alleged cases of sexual assault. Microscopic examination commonly used for spermatozoa identification is however time-consuming and can often lead to false-negative results for samples with deformed and, or, limited number of spermatozoa. To address this limitation, we report on a novel 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay to specifically identify spermatozoa. This assay is comprised of 3 markers: a digestive control marker (DC), sperm-specific marker (SP), and Y chromosome marker (SRY). A total of 214 samples from 10 body fluids or tissues were analyzed. Specificity testing showed that all the normal semen samples were unambiguously identified as being sperm-positive, and no other body fluid (or tissues) showed a sperm-specific signal in the electropherogram. Testing for sensitivity showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa by this assay. Mixture analyses illustrated the sensitivity of the assay when the vaginal/semen DNA ratio (80/0.1) was under 800 or the menstrual blood/semen DNA ratio (5/0.1) was under 50, the trace amounts (approximately 0.1 ng) of DNA from semen can still be identified by this 3-plex MSRE-PCR assay. This assay was also applied to the identification of 31 non-probative forensic samples from 18 sexual assault cases. The case studies showed that the 3-plex MSRE-PCR assay was an improvement in the sensitivity of spermatozoa detection.

Detection of cellular material in lip-prints.

We report on the visualization of cellular material within lip-prints using Diamond™ dye (DD). The transfer of cellular material via the lips can occur in cases of contact with food or drinking items as well as cases of alleged sexual assault involving oral contact. DD can effectively detect cellular material transferred by touch. Here we investigate if lip-prints can be detected and whether there is consistency within, or variability between, a person's propensity to shed cells within lip-prints. Ten volunteers were asked to press their lips against a glass slide with medium pressure for 15 s after not eating or drinking for at least 30 min. Both upper and lower lips were observed, and all tests were performed in five replicates, giving in total 900 observed areas. Consistency in the amount of cellular material deposited by lip-prints for each of the 10 individuals was observed, with each individual being associated with a 'lip shedder' status between the extremes of heavy and light. The majority of females shed more cells than the majority of males. No correlation was observed between the lip-prints shedder-status compared to deposition of cellular material from a thumb. Further, no correlation was observed between lip morphology and the 'lip shedder' status. Visualization of cellular material was not affected by lip-balm but was adversely affected by cosmetics such as lipstick. This technique demonstrates the visualization of deposited cells from parts of the body other than fingers and how cellular material can be visualized allowing targeted collection of DNA.

Establishment of 11 linked X-STR loci within 1.1 Mb to assist with kinship testing.

This report identifies and characterizes 10 novel short tandem repeat (STR) loci on the human X chromosome, all of which are within a range of 1.1 Mb. These newly characterized loci were developed to aid in kinship assignment when the X chromosome is specifically required. The repeat DNA sequences were identified initially using data in GenBank and are located immediately upstream and downstream from the previously described locus DXS6807. Only those loci with seven or more observed alleles were used for further study resulting in the identification of 10 new loci. The distance between each pair of loci ranged from 24,998 to 244,701 bp with an average of approximately 110.8 kb. The number of observed alleles ranged from 7 to 30 for these 10 loci with a polymorphic information content ranging from 0.593 to 0.930. The LOD score from a pairwise linkage study ranged from 4.40 to 23.73, indicating that these 11 loci were highly linked, as expected. In line with standard forensic practice, all 11 loci can be amplified in one multiplex reaction, and comprehensive allelic ladders for all the loci have been constructed. These newly established 11 linked STR loci on the human X chromosome were found to be highly polymorphic and have the potential to aid in kinship testing where the X chromosome loci currently plays a role.

A complementary forensic 'proteo-genomic' approach for the direct identification of biological fluid traces under fingernails.

Violent contact between individuals during a crime can result in body fluids becoming trapped under the fingernails of the individuals involved. The traces under fingernails represent valuable forensic evidence because DNA profiling can indicate from whom the trace originated and proteomic methods can be used to determine the type of fluid in the trace, thus providing evidence as to the circumstances surrounding the crime. Here, we present an initial study of an analytical strategy that involves two complementary techniques, direct PCR DNA profiling and direct mass spectrometry-based protein biomarker detection, for the comprehensive examination of traces of biological fluids gathered from underneath fingernails. With regard to protein biomarker detection, direct MALDI-ToF MS/MS is very sensitive, allowing results to be obtained from biological material present on only a few fibres plucked from a microswab used to collect the traces. Human cornulin, a protein biomarker for vaginal fluid, could be detected up to 5 h after it had been deposited under fingernails whereas haemoglobin, a biomarker for blood, is somewhat more persistent under fingernails and could be detected up to 18 h post-deposition. Bottom-up tandem mass spectrometry techniques were used to provide a high level of confidence in assigning the identity of protein biomarkers. nLC-ESI-qToF MS/MS offered higher levels of confidence and the ability to detect traces that had been present under fingernails for longer periods of time, but this performance came with the cost of longer analysis time and a more laborious sampling approach. Graphical abstract ᅟ.

The detection and identification of saliva in forensic samples by RT-LAMP.

We report on a novel method for saliva identification by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In our previous report, real-time RT-LAMP was used for blood identification by using HBB detection as a model but in this advanced study, this method was refined for the identification of the more challenging body fluid of saliva. Expression of the18S rRNA gene was used as the internal control and the Statherin (STATH) gene as the saliva-specific marker. A turbidimeter was used for real-time detection of the RT-LAMP products, and confirmation was obtained that the real products were generated using: agarose gel electrophoresis, calcein fluorescence detection and/or enzymatic digestion. The specificity of the test was performed using 42 samples including 7 different body fluids, and the expression of STATH was only observed in all the saliva samples (6) with a threshold time of 39.4 ± 2.9 min. Sensitivity testing showed that RT-LAMP products for STATH were stably detected when the RNA template was not less than 6.25 ng. When the primer concentrations for STATH were two times that of 18S rRNA, saliva could be identified in the body fluid mixtures even at a ratio (saliva:semen) of 1:3 (without loop primer)/1:5 (with loop primer). A multiplex RT-LAMP was established to simultaneously amplify the 18S rRNA and STATH genes, and applied to the identification of saliva on ten non-probative cigarette butts. A positive result for saliva was obtained from all ten butts, even for those that returned a negative or ambiguous result using the amylase test. A direct RT-LAMP test is also reported where the RNA extraction step was omitted to speed the collection of data and all tests using either the simplex or multiplex RT-LAMP resulted in a positive response if saliva was present. Our data provide a simple and effective means to detect the presence of saliva.

A mass spectrometry-based forensic toolbox for imaging and detecting biological fluid evidence in finger marks and fingernail scrapings.

During a crime, biological material such as blood or vaginal fluid may become smeared on the fingers of the victim or suspect or trapped under their fingernails. The type of trapped fluid is extremely valuable forensic information. Furthermore, if either person touches an object at the crime scene with their 'contaminated' finger then a 'contaminated' finger mark may be deposited. Such marks have great value as they could identify not only who deposited the mark but also who they touched and which part of the body they touched. Here, we describe preliminary work towards a 'toolbox' of techniques based on mass spectrometry (MS) for the identification of biological fluid traces under fingernails or the imaging of them in finger marks. Liquid chromatography-multidimensional MS was effective for the detection of protein biomarkers characteristic of vaginal fluid and blood trapped under fingernails, even after hands had been washed. In regard to examination of finger marks for the presence of biological fluids, the most practical implementation of any technique is to integrate it with, but after, routine crime scene finger mark enhancement has been applied. Here, we demonstrate the usage of matrix-assisted laser desorption ionization-time of flight-MS for the detection and mapping of proteins and peptides from body fluids in finger marks, including marks enhanced using aluminium-containing magnetic powder and then 'lifted' with adhesive tape. Hitherto, only small molecules have been detected in enhanced, lifted marks. In a novel development, aluminium in the enhancement powder assisted ionization of small molecules in finger marks to the extent that conventional matrix was not required for MS.

Ivory species identification using electrophoresis-based techniques.

Despite continuous conservation efforts by national and international organizations, the populations of the three extant elephant species are still dramatically declining due to the illegal trade in ivory leading to the killing of elephants. A requirement to aid investigations and prosecutions is the accurate identification of the elephant species from which the ivory was removed. We report on the development of the first fully validated multiplex PCR-electrophoresis assay for ivory DNA analysis that can be used as a screening or confirmatory test. SNPs from the NADH dehydrogenase 5 and cytochrome b gene loci were identified and used in the development of the assay. The three extant elephant species could be identified based on three peaks/bands. Elephas maximus exhibited two distinct PCR fragments at approximate 129 and 381 bp; Loxodonta cyclotis showed two PCR fragments at 89 and 129 bp; and Loxodonta africana showed a single fragment of 129 bp. The assay correctly identified the elephant species using all 113 ivory and blood samples used in this report. We also report on the high sensitivity and specificity of the assay. All single-blinded samples were correctly classified, which demonstrated the assay's ability to be used for real casework. In addition, the assay could be used in conjunction with the technique of direct amplification. We propose that the test will benefit wildlife forensic laboratories and aid in the transition to the criminal justice system.

Investigation into length heteroplasmy in the mitochondrial DNA control region after treatment with bisulfite.

We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.

DNA profiles from clothing fibers using direct PCR.

We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number.

Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system.

PURPOSE: Knowledge of the composition of complex body fluid mixtures may aid forensic investigations greatly. However, many of the traditional tests are presumptive in nature and can lead to ambiguous results. The aim of this study is to establish a reliable method to identify various biofluids via analysis of their DNA methylation profiles. METHODS: A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids, and semen were isolated from the open database of Infinium HumanMethylation450 BeadChip. These biofluid-specific markers, a control marker to confirm bisulfite conversion, and a gender marker, were combined into a 10-plex methylation-specific PCR single-base-extension (MSP-SBE) system. RESULTS: Analysis of 65 DNA samples isolated from venous blood, semen, vaginal fluid, saliva, and menstrual blood that had been treated with bisulfite, resulted in all eight markers detecting the body fluid to which they were designed. Unambiguous body fluid identification occurred from both single sources of body fluids and complex mixtures. A threshold was devised for each marker to minimize the chance of a false inclusion. The efficacy of the assay and application to forensic practice was demonstrated using five non-probative samples from real alleged sexual assault cases. The system unambiguously determined the biofluid types for the non-probative forensic samples that previously resulted in inconclusive or conflicting results using traditional tests. CONCLUSIONS: The results demonstrated the 10-plex MSP-SBE system established in this study is both sensitive and specific when applied to body fluid identification and can be readily adopted into forensic practice.