Comparison of DNA extraction methods for identification of human remains

After mass disasters, where the bodies of victims have been degraded, soft tissues are often completely lost, with the result that bones and teeth are all that remain for identification. The aim of this study was to investigate three DNA extraction methods that are currently used with degraded bones in forensic contexts: a silica-based extraction protocol used by the International Commission for Missing Persons (ICMP), a total demineralisation method used by the US Armed Forces DNA Identification Laboratory (AFDIL) and a standard organic DNA extraction method used by the Australian Federal Police (AFP). The methods were compared by real-time PCR quantitation and STR profiling. The silica and organic methods were not significantly different for either of these measures for bone but were both significantly more successful than the demineralisation method. Bovine serum albumin (BSA) was found to significantly improve the DNA yields from real-time PCR and the number of reportable alleles from STR typing, especially when using bone samples.     

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store.We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA^® cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor^®HY. All samples were amplified using PowerPlex^®21 and PowerPlexY^®23 systems and analyzed using the AB3500 Genetic Analyzer and the GeneMapper^® ID-X v.1.2 software.PCR inhibitors were consistently detected in bone marrow, muscle tissue, rib and vertebra samples. Amplifiable DNA was obtained in a majority of the samples analyzed. DNA recovery from 0.1 g biological material was adequate for successful genotyping of most of the non-bone and bone samples. Complete DNA profiles were generated from bone marrow, femur, metatarsal and patella with 0.1 ng DNA template. Using 0.5 ng DNA template resulted in increased allele recovery and improved intra- and inter-locus peak balance.     

Post-blast detection of human DNA on improvised explosive device fragments

ABSTRACT

Many terrorist attacks employ the use of improvised explosive devices such as pipe bombs. In these circumstances, the perpetrator may be absent from the scene, necessitating the need for a quick resolution. Forensic DNA analysis is one of the key disciplines utilized to identify possible offenders in terror-related crimes; however, its success in post-blast environments is unpredictable. Through using a known quantity of human DNA on pre-blast samples, post-blast DNA results can be assessed for STR profiling suitability. In this study, two pipe bombs were constructed and doped with varying concentrations of human blood. The samples were extracted using the DNA IQ System extraction kit and quantified using the Quantifiler Trio DNA Quantification Kit. The results obtained from this analysis confirm the presence of DNA post-blast. The variation between post-blast and pre-blast samples was not found to be statistically significantly different. Furthermore, the results indicate that 55% of the samples quantified post-blast could produce partial or full profiles in downstream DNA testing.     

A law enforcement intelligence framework for use in predictive DNA phenotyping

ABSTRACT

Analysis of information about physical characteristics, biogeographical ancestry or common genetic ancestors from crime scene DNA is a technique aimed at informing an intelligence process, rather than obtaining evidence for a criminal trial. This intelligence supports tactical or operational decision-making. Like other forms of intelligence there is a risk for it to be misconstrued or for its investigative value to be misunderstood. The potential for intelligence derived from DNA to divert investigative resources or result in unnecessary intrusions into individual privacy can be mitigated by applying an appropriate intelligence doctrine. Establishing an appropriate framework could reduce the need for government regulation of these emerging capabilities in the context of law enforcement use.     

Evaluation of DNA profiles obtained from deceased individuals at Salt River Mortuary (South Africa)

ABSTRACT

DNA profiling is a valuable tool for human identification, particularly when deceased individuals cannot be identified by traditional methods. Annually, at Salt River Mortuary (SRM), ~320 bodies remain unidentified following post-mortem investigation. While much research has been conducted into DNA profiling on living individuals, relatively little has focused on suitability within the deceased population, and thus this formed the focus of this research. Buccal cells were collected from deceased infants (n = 38) and adults (n = 37) at SRM, with a time interval between death and sample collection of 1 – 887 days. Control blood samples were obtained from a subset of participants. Samples were processed using the PowerPlex ESI 16 System or Y23 System (Promega). Full DNA profiles were obtained from all blood samples. Following optimization, 74.7% and 18.7% of buccal swabs yielded full and partial profiles respectively, while 6.7% failed completely. Upon analysis, partial and failed profiles were significantly associated with the DNA degradation index from cotton swabs (p < 0.001), but not with time between death and sample collection (p = 0.16). These results indicate that full DNA profiles can be obtained long after death. It is hypothesized that factors influencing degradation (e.g. decomposition) may contribute to failure. Overall, further research is required to identify factors influencing DNA profile quality.     

Presentation Of DNA Data in Cases of Disputed Paternity

Flip-open style cell phones were investigated for the potential to produce quality genetic profiles that could be used in forensic casework. Swabs were taken of the outside/back and the inside ear speaker of ten flip-phones on two occasions – prior to and seven days after cleaning with 95% ethanol. Buccal swabs were collected as exemplars. The samples were amplified using the AmpFlSTR ProfilerPlus PCR Kit for 35 cycles and STR profiles were generated using an ABI Prism 310 Genetic Analyzer and GeneMapper ID analysis software v3.2. The phone profiles were compared to the references and to each other, to assess the quality of the profiles. The completeness of the profiles varied greatly, even within an experimental condition. There was no significant difference in the percentage correct alleles or in the number of drop-in alleles in the DNA profiles for the outside/inside locations or for the pre/post-cleaning times. The findings of this study demonstrate the need for collecting multiple samples from discrete locations on a cell phone if such evidence is encountered in forensic cases.     

A forensically validated genetic toolkit for the species and lineage identification of the highly trafficked shingleback lizard (Tiliqua rugosa)

Shingleback lizards (Tiliqua rugosa) are among the most trafficked native fauna from Australia in the illegal pet trade. There are four morphologically recognised subspecies of shinglebacks, all with differing overseas market values. Shinglebacks from different geographic locales are often trafficked and housed together, which may complicate identifying the State jurisdiction where the poaching event occurred. Additionally, shinglebacks can be housed and trafficked with other species within the same genus, which may complicate DNA analysis, especially in scenarios where indirect evidence (e.g. swabs, faeces) is taken for analysis. In this study, a forensic genetic toolkit was designed and validated to target shingleback DNA for species identification and geographic origin. To do this, field sampling across Australia was conducted to expand the phylogeographic sampling of shinglebacks across their species range and include populations suspected to be poaching hotspots. A commonly used universal reptile primer set (ND4/LEU) was then validated for use in forensic casework related to the genus Tiliqua. Two additional ND4 primer sets were designed and validated. The first primer set was designed and demonstrated to preferentially amplify an ∼510 bp region of the genus Tiliqua over other reptiles and builds on existing data to expand the available phylogeographic database. The second primer set was designed and demonstrated to solely amplify an ∼220 bp region of T. rugosa ND4 over any other reptile species. Through the validation process, all primers were demonstrated to amplify T. rugosa DNA from a variety of sample types (e.g. degraded, low quality and mixed). Two of the primer sets were able to distinguish the genetic lineage of T. rugosa from the phylogeographic database. This work provides the first forensically validated toolkit and phylogeographic genetic database for Squatmate lizards.     

Molecular approaches for forensic cell type identification: On mRNA,miRNA, DNA methylation and microbial markers

Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well.     

Evaluation of a personal identification method using the fusion function of CT images and dental radiographs

Objectives:

We applied the fusion function of dental analysis software to examine whether the anatomical relationships of tooth roots when compared between reconstructed post-mortem CT (PMCT) and dental radiographs can aid dental identification.

Methods:

One PMCT image taken from a cadaver (43-year-old male; Cadaver 1) was compared with 64 digital dental radiographs of the left and right upper and lower molars from Cadaver 1 and 30 other cadavers. Five corresponding anatomical reference points were marked on each image. After adjusting the angle and magnification using the fusion function, the automatically calculated error in pixels was determined five times for each of four sites on the images.

Results:

Comparison of the PMCT image with the dental radiographs from the other cadavers revealed obvious discrepancies in the anatomical positioning of the teeth. When t-tests were applied to the data from any of the four sites, the error in pixels was found to be significantly smaller (p < 0.001) between Cadaver 1 and the other cadaver images. The average error in pixels between the PMCT and dental radiographs was smaller in the lower jaw than in the upper jaw.

Conclusions:

This method uses corresponding reference points on two images to calculate the error between the regions that contain all points. This feature also makes it possible to compare images taken with different modalities. The demand for a dental identification method involving PMCT is likely to increase, and we expect that the accuracy of dental identification will improve by using radiological images.     

An analysis of the success rate of 908 trace DNA samples submitted to the Crime Sample Database Unit in New Zealand

The Crime Sample Database Unit, part of the Forensic Biology group at the Institute of Environmental Science and Research Limited, receives samples predominantly from unsolved volume crime cases, such as burglaries and car theft, for inclusion on the New Zealand National DNA Databank. A high proportion of these samples are analysed for the presence of low or trace amounts of DNA. A study was undertaken to evaluate the success of such sample types in providing a DNA profile. Handled items expected to yield low or trace levels of DNA were found to be a commonly submitted type of item in this group, but one of the least successful in terms of DNA profiles obtained. These results are significant, as much time and money is invested in the analysis of such samples, resources that may be better employed elsewhere.     

Development of a SNP-based panel for human identification for Indian populations

The widely employed short tandem repeat (STR)-based panels for forensic human identification (HID) have limitations while dealing with challenging forensic samples involving DNA degradation, resulting in dropping-out of higher molecular weight alleles/loci. To address this issue, bialleic markers like single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), which can be scored even when the template DNA is heavily degraded (<100 bp), have been suggested as alternative markers for HID testing. Recent studies have highlighted their utility in forensic HID and several panels based on biallelic markers have been described for worldwide populations. However, there has been very little information about the behavior of such DNA markers in Indian populations, which is known to possess great genetic diversity. This study describes a two-step approach for designing a SNP-based panel consisting of 70 SNPs for HID testing in Indian populations. In the first step, candidate SNPs were shortlisted from public databases by screening them for several criteria including allelic distribution, genomic location, potential phenotypic expression or functionality and species specificity. The second step involved genotyping the shortlisted SNPs in various Indian populations followed by shortlisting of the best performers for identity-testing. Starting with 592,652 SNPs listed in Human660W-Quad Beadchip (Illumina Inc.), we shortlisted 275 candidate SNPs for identity-testing and genotyped them in 462 unrelated individuals from different population groups in India. Post genotyping and statistical analyses based on biogeographic regions, 206 SNPs demonstrated desired allelic distribution (Heterozygosity ≥ 0.4 and F_ST ≤ 0.02), from which 2–4 widely separated (>20 Mb apart) SNPs from each chromosome were finally selected to construct a panel of 70 SNPs. This panel on average possessed match probability 10e-29 and probability of paternity of 0.99999997, which was orders of magnitude higher than most of the currently employed STR-based chemistries and SNP-based panels that were proposed previously for HID testing. For comparison purpose, genotyping previously reported SNPs for HID in our samples led us to conclude that the panel developed in this study is much more efficient and robust and better suited for the Indian populations.     

Molecular studies of time- and environment-dependent effects on bone DNA survival

In the past few years, many studies of human identification through DNA analysis of skeletal material have been published; however, the potential use of DNA technology to determine the postmortem interval (PMI) is still under investigation. Compared with soft tissue, skeletal materials provide longer protection against DNA degradation. We examined the effects of PMI and environmental conditions, i.e. temperature and humidity on bone DNA survival by examining human rib bone samples. Samples were taken within 24 hours postmortem and then stored from 103 to 445 days in four different conditions, namely freezing, ambient conditions, high humidity and underground burial. Bone DNA extracts were assessed by gel electrophoresis. The authenticity of the DNA could be verified by Alu sequence Southern blot hybridization. The results confirmed the strong effects of environment on DNA stability. Burial and high humidity conditions resulted in almost complete degradation of DNA in our samples. However, there was no correlation detected between the amount of DNA or the pattern of DNA fragmentation and PMI in our studies.     

Development and validation of a fast and automated DNA identification line

The importance of DNA evidence for gaining investigative leads demands a fast workflow for forensic DNA profiling performed in large volumes. Therefore, we developed software solutions for automated DNA profile analysis, contamination check, major donor inference, DNA database (DDB) comparison and reporting of the conclusions. This represents the Fast DNA IDentification Line (FIDL) and this study describes its development, validation and implementation in criminal casework at the authors’ institute. This first implementation regards single donor profiles and major contributors to mixtures. The validation included testing of the software components on their own and examination of the performance of different DDB search strategies. Furthermore, end-to-end testing was performed under three conditions: (1) testing of scenarios that can occur in DNA casework practice, (2) tests using three months of previous casework data, and (3) testing in a casework production environment in parallel to standard casework practices. The same DNA database candidates were retrieved by this automated line as by the manual workflow. The data flow was correct, results were reproducible and robust, results requiring manual analysis were correctly flagged, and reported results were as expected. Overall, we found FIDL valid for use in casework practice in our institute. The results from FIDL are automatically reported within three working days from receiving the trace sample. This includes the time needed for registration of the case, DNA extraction, quantification, polymerase chain reaction and capillary electrophoresis. FIDL itself takes less than two hours from intake of the raw CE data to reporting. Reported conclusions are one of five options: (1) candidate retrieved from DDB, (2) no candidate retrieved from DDB, (3) high evidential value with regards to reference within the case, (4) results require examination of expert, or (5) insufficient amount of DNA obtained to generate a DNA profile. In our current process, the automated report is sent within three working days and a complete report, with confirmation of the FIDL results, and signed by a reporting officer is sent at a later time. The signed report may include additional analyses regarding e.g. minor contributors. The automated report with first case results is quickly available to the police enabling them to act upon the DNA results prior to receiving the full DNA report. This line enables a uniform and efficient manner of handling large numbers of traces and cases and provides high value investigative leads in the early stages of the investigation.     

A forensic case study for body fluid identification using DNA methylation analysis

Recently, a method of identifying body fluids using DNA methylation has been developed (Frumkin et al., 2011). An existing multiplex assay using 9 CpG markers could differentiate 5 body fluids: semen, blood, saliva, menstrual blood, and vaginal fluid. To validate this technique, we evaluated the previously described body fluid identification method by means of single base extension (SBE). DNA methylation was applied to 22 samples in 18 forensic cases; seven of these were semen, three were blood, eight were saliva, three were vaginal fluid, and one was menstrual blood. Total of 18 samples were tested, the DNA methylation profiles were coincident from preliminary tests (acid phosphatase (AP), leucomalachite green (LMG, Sigma Aldrich, St Louis, MO, USA) and SALIgAE®) except one sample which displayed an all-negative result. After applying the DNA methylation method to forensic samples, we determined that it could be very useful for differentiating vaginal secretions from menstrual blood, for which there is no conventional preliminary testing method.     

Establishing historical sample data is essential for identification of unaccounted Australian soldiers from WWI,WWII, and the Korean War

ABSTRACT

Mitochondrial DNA (mtDNA) is used for identification of Australian military personnel whose remains are recovered from historical conflicts. A mtDNA sample database does not exist for Australian soldiers that served in World War I (WWI), World War II (WWII) and the Korean War, meaning it is unknown what common haplotypes may have existed among these soldiers, risking identification errors. Haplotype diversity (position 16,024 to 548) was examined in a sample of 254 unrelated WWII-era European-Australians. Of these, 220 different haplotypes were observed, and it is estimated that between 18% and 29% of Australian soldiers who served in historical conflicts have common haplotypes (95% CI). This research demonstrates that mtDNA control region analysis of historical military remains will provide a lower than expected power of discrimination given the population structure of the time, and enlistment policies targeting Australians of European decent. The point estimates for 52% of the common haplotypes obtained in the historical European-Australian sample were not represented in the confidence intervals for European and Western-European EMPOP data. Creation of targeted sample data reflecting correct ancestry of the WWI and II soldiers and additional mtDNA and Y-STR analysis are essential to avoid misidentification of Australian soldiers from historical conflicts.     

Validation study of a 15-plex rapid STR amplification system for human identification

Conventional PCR amplification requires approximately 3 h to complete and thus does not meet the requirements of rapid DNA analysis. The purpose of this study was to validate a rapid 15-plex PCR system that can amplify 14 autosomal short tandem repeat (STR) loci (i.e., D6S1043, D21S11, D7S820, CFS1PO, D2S1338, D3S1358, D13S317, D8S1179, D16S539, Penta E, D5S818, vWA, D18S51, and FGA) and Amelogenin. This system was validated by sensitivity, species specificity, inhibitor tests, sizing accuracy, stutter calculation, concordance tests, DNA mixture, and case sample tests according to the Scientific Working Group for DNA Analysis Methods (SWGDAM) guidelines and Chinese criteria. We found that the rapid 15-plex PCR system could shorten the amplification time to 37 min and proved that it provides an alternative method for conventional PCR in human identification detection.     

Evaluation of a co-extraction method for real-time PCR-based body fluid identification and DNA typing

Body fluid identification and individual identification are an important series of tests in usual criminal investigations. Recent reports have demonstrated a new approach using DNA/RNA co-extraction methods in which RNA for body fluid identification and DNA for short tandem repeat (STR) typing are extracted simultaneously from the same sample. This study evaluated a standard co-extraction kit, the AllPrep® DNA/RNA Mini Kit, in order to demonstrate the availability of the co-extraction procedure for those real-time polymerase chain reaction-based body fluid identification methods that we have validated previously. We demonstrated that the use of the Allprep Kit, for which we adjusted the lysis temperature to 56 °C to improve extraction efficiency, can simultaneously extract sufficient RNA and DNA for body fluid identification and STR analysis; however, a longer incubation at a high temperature slightly affected the ΔCt value of each target gene and appeared to be not as effective for DNA extraction from old stains as from 1-day-old stains. This method is promising for future forensic investigations because the use of this kit can reduce sample consumption for body fluid identification and DNA typing.     

Forensic Science in a Human Rights Framework

Forensic services, founded upon good science and best practice, provide an inherent safeguard for human rights. Moreover, practitioners are well placed to uphold fundamental and longstanding rights such as ‘the right to a fair hearing’. Our ability to embrace other emerging rights, however, is less clear. The increasing ambit and remit of forensic science is a cause of increasing social, legal and ethical concerns. Furthermore, there is an inherent tension between the notion of ‘value free scientific method’ and the conflicting dictates of government policies, which seek to recognise and protect greater religious, ethnic and cultural freedoms whilst being increasingly reliant upon forensic science in the detection, prevention and prosecution of criminality.

This paper addresses, from the perspective of a practitioner, the complex relationship between forensic science and society and the implications of new developments upon our role in the criminal justice system within a human rights framework. In particular, this paper argues that forensic practitioners must actively engage with societal concerns. We cannot be simple onlookers in this debate.     

Ribosomal DNA as target for the assessment of DNA degradation of human and canine DNA

The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing success is often hampered by low amounts and strong degradation of nuclear DNA. Common strategies consist of quantitative polymerase chain reaction (qPCR)-based analysis of the abundance of a short versus a long nuclear amplicon, the latter prone to DNA degradation. To increase sensitivity, commercial qPCR solutions rest on amplification of multi-copy DNA sequences. Here we show that ribosomal DNA (rDNA) sequences are well suited for the same purpose. Because rDNA sequences are present in high copy number in most eukaryotic species, qPCR strategies can easily be adapted to non-human species. In this paper, we establish qPCR-based assays for human or dog DNA, respectively, which allow for sensitive analysis of DNA amounts and DNA degradation. We show that the human system can be applied to DNA of single telogen hairs, where STR typing success correlates with measured amounts and integrity of the DNA. By adapting the system to dog rDNA sequences we found that single telogen dog hairs often displayed less DNA degradation than human telogen hairs, in most cases allowing for successful STR typing. Thus, qPCR-based analysis of rDNA represents a cost-effective, highly sensitive strategy to assess DNA amount and integrity that can be adapted to hairs or other traces from various animal species.     

Evaluation of a co-extraction kit for mRNA,miRNA and DNA methylation-based body fluid identification

Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.