Forensic human identification: Investigation into tooth morphotype and DNA extraction methods from teeth

When a body is decomposed, hard tissues such as teeth may provide the only DNA source for human identification. There is currently no consensus as to the best DNA extraction method, and there is a lack of empirical data regarding tooth morphotype and condition that may impact DNA recovery. Therefore, this study sought to investigate which variables significantly improved DNA concentration, integrity and profiling success. A total of 52 human teeth were assessed, representing all tooth morphotypes from three deceased individuals. DNA was extracted using both the QIAamp® DNA Investigator Kit and the phenol-chloroform method. DNA concentration and degradation index were assessed using real time PCR, prior to conventional DNA profiling. Contrary to international guidelines promoting the use of molars, DNA profiling from molars was the least successful, with premolars, followed by canines, performing the best. The presence of fillings reduced the DNA quantity and quality obtained and may explain the poor performance of molars. DNA from the maxillae were significantly less degraded when the QIAamp® was used, although this did not influence DNA profiling success. A significant increase in DNA concentration, integrity and profiling success was observed in diseased teeth (periodontitis) compared to those without disease. This may be due to increased white blood cell presence at the site. There was no significant difference in DNA profiling success between the two DNA extraction methods. However, different teeth yielded failed DNA profiles for each extraction method, suggesting that repeated attempts, using alternative DNA extraction methods, is recommended. The recovery of additional DNA profiling information from degraded samples may help to ultimately reduce the burden of unidentified human remains.     

Personal identification of cold case remains through combined contribution from anthropological, mtDNA, and bomb-pulse dating analyses

In 1968, a child's cranium was recovered from the banks of a northern Canadian river and held in a trust until the "cold case" was reopened in 2005. The cranium underwent reanalysis at the Centre for Forensic Research, Simon Fraser University, using recently developed anthropological analysis, "bomb-pulse" radiocarbon analysis, and forensic DNA techniques. Craniometrics, skeletal ossification, and dental formation indicated an age-at-death of 4.4 ± 1 year. Radiocarbon analysis of enamel from two teeth indicated a year of birth between 1958 and 1962. Forensic DNA analysis indicated the child was a male, and the obtained mitochondrial profile matched a living maternal relative to the presumed missing child. These multidisciplinary analyses resulted in a legal identification 41 years after the discovery of the remains, highlighting the enormous potential of combining radiocarbon analysis with anthropological and mtDNA analyses in producing confident personal identifications for forensic cold cases dating to within the last 60 years.     

Quantifiler Human DNA Quantification Kit (Applied Biosystems) as a screening kit for DNA profiling

This study investigates the connection between the results of DNA quantification with Quantifiler Human DNA Quantification Kit (AB) and DNA profiling. For this purpose the DNA concentration of 3.068 routine casework samples was determined and DNA profiling was carried out. For discussion, depending on the specific DNA concentration, the samples were divided into four groups (0–5, 5–10, 10–30 and more then 30 pg/μl DNA) and the obtained number of full or partial profiles and the negative typing results was listed. Moreover group 1 (0–5 pg/μl DNA) results were subdivided and analysed more precisely. Based on the amount of 4% positive typing results in group 1 we decided to analyse every sample with quantification results >0 pg/μl DNA. A real cut-off no longer exists. Only samples showing 0 pg/μl on each result were sorted out.     

STR and SNP analysis of human DNA from Lucilia sericata larvae's gut contents

Importance of forensic entomology becomes inevitable when come across some incident where corpse is unidentifiable and lot of maggots or other insects are present. The most common application of forensic entomology is to use insects for the identification of specimens or human remains. DNA analysis recovered from a larva's gut contents can be used to identify a missing body. The obtained human STR and SNP profile support the association of a maggot to a specific patients or corpse. Main aim of this research was the identification of human DNA from gut contents of third instar maggots (larvae of Lucilia sericata) placed on diabetic patient's wounds for treatment purpose. Maggots (8–15) were taken from each diabetic patients (no. of the patients 8) and DNA was extracted from the gut contents manually by using Qiagen tissue protocol. Agarose gel electrophoresis was performed and the total size of DNA was seen using UV transilluminator. PCR amplification, STRs and SNPs profiling was then performed using PCR 9700 and AmpFLSTR Identifiler and SnaPshot Multiplex Kit (Applied Biosystems) respectively. The results were analyzed on ABI 310. SNP profiles were good and identifiable compared to the STRs where amplification was poor and the peaks were low. This may be the fact of the enzymatic activity present in the gut of the larvae which cause tremendous reduction in DNA size and thus yield. The results of this study reveals that it is possible to obtain a complete human profile using STRs and SNPs even if DNA is recovered from gut contents of maggots.     

A fast and safe non-bleaching method for forensic skeletal preparation

Over the last three decades, forensic anthropologists increasingly have consulted on fleshed human remains cases in which the examination of skeletal elements is critical in answering questions of identification and the circumstances of death. This was certainly the case at the Human Identification Laboratory in Tucson, Arizona. As the caseload increased, it became clear that a method for defleshing human remains was needed in order to expeditiously expose the osseous surfaces for analysis, yet at the same time, preserving the evidentiary nature of the material. As a result, a fast, safe and economical method for defleshing human remains and producing high quality, degreased skeletal elements was developed. This non-bleaching cooking method utilizes chemicals that are easily obtained and inexpensive standard household ingredients that can be purchased at most grocery stores.     

Anthropological and Radiographic Comparison of Antemortem Surgical Records for Identification of Skeletal Remains*

Abstract: This case review illustrates the important contributions of forensic archeological methods and forensic anthropological analysis to the identification of found skeletal remains. After reassociation of skeletal remains found in two locations, anthropological analysis provided the basis for a presumptive identification and a request for antemortem medical records. Partial DNA profiles were supportive but not conclusive and antemortem dental records were not available. Comparison of antemortem traumas, skeletal morphology, and surgical artifacts with antemortem radiographs and surgical records led to positive identification of an individual missing for almost a decade.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

Human being eaten by his own dogs: Genetic confirmation through analysis of bones recovered in a dog's stomach content

A part of a decomposed human body from an individual was found at his home, together with the decomposed bodies of his 3 dogs. The disappearance of half of the human body was hardly explained. Hypothetically the dogs, starving, could have eaten their owner after his death. All the bodies were recovered for autopsy.Small bone fragments were recovered in one dog's stomach and identified as human by anthropological analysis. DNA was extracted and cytochrome b gene analysis was made in order to determine their origin, confirmed as human.Genetic identification allowed achieving an eight mini-STR profile with MiniFiler (Applied Biosystems) identical to the bone material collected at the victim's autopsy, confirmed that the dogs had effectively eaten their owner.The results showed that it is possible to obtain nuclear DNA in samples subjected to gastric acids and the combination of different techniques allowed us to determine, in each step, the most convenient workflow for the remains identification.     

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection – one per each body fluid type – demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.     

DNA profiling of disaster victim identification in Trenggalek shipwreck case

DNA analysis is one of the primary methods of identification in DVI practices. The external environment of a mass disaster often results in severe fragmentation, decomposition and intermixing of the remains. However, DNA profiling still can be achieved even on cases involving partial, severely decomposed remains. This report shows the DNA profile of shipwreck victims using identifiler plus marker from tissue sample exposed to environmental conditions.     

Symmetrical fracturing of the skull from midline contact gunshot wounds: reconstruction of individual death histories from skeletonized human remains

This paper reports a bilaterally symmetrical cranio-facial fracture pattern that is observed in self-inflicted, midline gunshot wounds. Five cases of self-inflicted gunshots wounds are presented as follows: two high-powered rifle cases, two shotgun cases, and one handgun case. In all five cases the remains were either decomposing or skeletonized and submitted to forensic anthropologists. Following identification, the main focus of the anthropological examination was the analysis of perimortem trauma to the skeleton. In each case, the skull was submitted in a highly fragmented state. Nevertheless, by focusing on the pattern of perimortem cranio-facial fractures, the anthropologists contributed key information regarding the circumstances of death. The observed symmetrical cranio-facial fracture patterns in the above cases are described in detail and interpreted. The specific location of the linear fractures is discussed, as well as the theoretical rationale behind the location in terms of skeletal architecture, such as buttresses, struts, and sutures. The interpretive framework provided by this paper may prove helpful to others who are faced with similar cases of cranio-facial fracturing.     

A qualitative study of compact bone microstructure and nuclear short tandem repeat obtained from femur of human remains found on the ground and exhumed 3 years after death

Forensic identification of human remains is composed of anthropological study of race, sex, age, etc. By using these traditional methods, inconclusive or nonidentified cases could be subjected to DNA analysis. However, in spite of advances in human identification techniques, especially by PCR-amplified DNA, some limitations that affect the ability of obtaining DNA from human remains still persist. Light microscope sections of postmortem compact bones from human remains are presented here for the purpose of increasing a forensic examiner's prediction of successful nuclear DNA typing. Femoral compact bones were obtained from 7 human remains found on the ground, in different degrees of decomposition, and were cleaned by boiling to remove soft tissues, 8 collections of bones having undergone natural decomposition, not boiled (as no soft tissue was adhered), and 5 cadavers 12 to 16 hours postmortem. The histologic sections were stained by hematoxylin and eosin, the loci CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, and amelogenin were amplified by PCR, and the polyacrylamide gel was stained with silver. The results presented here clarify questions concerning the viability of DNA for identification analysis, and they also may help to establish better strategies for optimization of DNA extraction and analysis in compact bones of human remains.     

DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala

No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.     

Identification of decomposed human remains from radiographic comparisons of an unusual foot deformity

A case of positive identification from decomposed human remains using an unusual foot deformity is presented. Scrutiny of the decedent revealed foot deformities, which upon examination, prompted further inquiry. Radiographic comparisons and defleshing each foot established bilateral talipes equinovarus (TEV, clubfoot). Positive identification was based upon unique skeletal features present in the radiographs.     

Teeth as a source of DNA for forensic identification of human remains: A Review

Teeth and bones are frequently the only sources of DNA available for identification of degraded or fragmented human remains. The unique composition of teeth and their location in the jawbone provide additional protection to DNA compared to bones making them a preferred source of DNA in many cases. Despite this, post-mortem changes in the structure and composition of teeth, and the location and diagenesis of DNA within them are poorly understood. This review summarises current knowledge of tooth morphology with respect to DNA content and preservation, and discusses the way in which post-mortem changes will affect the recovery of DNA from teeth under a range of commonly used extraction protocols. We highlight the benefits and pitfalls of using specific tooth tissues for DNA extraction and make recommendations for tooth selection and sampling that will maximise DNA typing success. A comprehensive understanding of tooth structure and an appreciation of the relationship between DNA and mineralized tissues in post-mortem teeth are critical for optimal sample selection. More informed sampling methods that target specific tooth tissues will increase the likelihood of successful genetic analysis and allow for efficient and timely missing persons case work and disaster victim identification response.     

DNA extraction of burnt bone and teeth casework samples using bead-beating homogenization technique

Sample disruption was a necessary step for DNA isolation. Bone and teeth were useful biological sources particular in human remains and advance decomposed bodies. The compact bone and teeth required several preparation steps prior to analyzing process. However, the methods in standard protocol were laborious and time consuming. An alternating pulverization, bead beating homogenizer, was purposed in its effectiveness for forensic casework. (1) Here, we applied this technique to the burnt cracked bone and tooth that recovered from house fire for forensic DNA analysis. After cleansed an external surface, the eight multidirectional motion tissue homogenizer, Precellys® evolution, was utilized to pulverize bone and tooth followed by a DNA extraction and amplification. For detection with a capillary electrophoresis, full profiles of autosomal STRs and Y-chromosomal STRs were recovered from tooth sample but the partial profile STR was demonstrated in bone sample. The new technique in bone homogenization was less time consuming (around 30 s), less exposure to chemical agents (no need of liquid nitrogen), high efficiency, with high-throughput productivity.     

Different skeletal elements as a source of DNA for genetic identification of Second World War victims

To this day process of identification of missing persons from skeletonized human remains with help of forensic genetics proves to be complex and challenging. The success rate of genetic identification in bones strongly depends on a combination of various factors, most importantly environmental factors and post-mortem interval. Furthermore, there are individual-specific factors that affect DNA preservation, such as race, gender, age and type of skeletal elements. The goal of our study was to optimize sampling process through determining which skeletal elements are superior in their preservation of DNA in 70-yearold skeletons belonging to victims of Second World War. We sampled different types of bones and teeth from three such skeletons found in Slovenian hidden mass grave Huda jama, 56 elements from each respective skeleton, together 168 elements. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Prior to powdering bones and teeth, we removed contaminants. We decalcified 0.5 g bone and tooth powder followed by extraction and purification of DNA using Biorobot EZ1 (Qiagen). Quantification of obtained nuclear DNA was carried out using PowerQuant kit (Promega) and autosomal STR typing using ESSplex SE QS kit (Qiagen). Best parameters to assess skeletal elements that are superior in their DNA preservation were quantity of DNA and number of successfully typed STR loci. Metacarpal and metatarsal bones proved to be the best, followed by intermediate cuneiform, first distal foot phalanx, talus, petrous bone and tibia. We also created elimination database for persons involved in exhumation, anthropological and genetic analyses and exclude potential contamination.     

Validation studies of the ParaDNA® Intelligence System with artificial evidence items

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA^® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.