Forensic DNA Challenges: Replacing Numbers with Names of Fosse Ardeatine’s Victims*

Abstract: The Fosse Ardeatine massacre was a mass execution carried out in Rome on March 24, 1944 by Nazi German occupation troops during the Second World War as a reprisal for a partisan attack conducted on the previous day in central Rome. The 335 civilians were taken to the “Cave Ardeatine” and they were shot. Only 323 corpses out of 335 have been identified. The aim of this work is the genetic and anthropological analysis of the remains exhumed from grave number 329 of Fosse Ardeatine’s Shrine to assess their identity. So far, such remains have been supposed to belong to MM but mitochondrial analysis excluded a biological relationship to two living maternal relatives. Our analysis indicated that remains recovered in grave number 329 do not belong to MM. This result suggests that genetic analysis of the remains should be also applied to the other 12 unknown corpses to elucidate their identity.     

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

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

荧光原位杂交技术在法庭科学DNA检验中的应用

目的运用荧光原位杂交技术结合激光显微切割技术,分离法医物证男女混合样本中的男性细胞和女性细胞,并进行DNA分型。方法通过双色荧光原位杂交,Y染色体标记上绿色信号,X染色体标记上红色信号,在荧光显微镜下识别男性细胞和女性细胞,并通过激光显微切割技术分别获得男性细胞和女性细胞进行DNA分型。结果运用荧光原位杂交技术,能够分别标记法医物证男女混合样本中的男性细胞和女性细胞,并通过激光显微切割技术获得各自的DNA分型。结论荧光原位杂交技术结合激光显微切割技术,可应用于法医物证男女混合样本的检验,提高个体识别能力。     

A Grass Molecular Identification System for Forensic Botany: A Critical Evaluation of the Strengths and Limitations*

Abstract: Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA‐based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single‐nucleotide polymorphisms were utilized as molecular markers for subfamily to species‐level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated “proof of concept” of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.     

Molecular Identification of Indian Crocodile Species: PCR‐RFLP Method for Forensic Authentication*

Abstract: South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile‐based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA‐based identification of species using PCR‐RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species‐specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.     

DNA Barcoding in Forensic Entomology – Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species,

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High‐quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1‐5P’ DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high‐quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

The Conviction of Dr. Crippen: New Forensic Findings in a Century‐Old Murder

Abstract: Dr. Hawley Crippen was accused and convicted of murdering his wife in London in 1910. Key to the conviction was microscopic analysis of remains found in the Crippen’s coal cellar, which were identified as Cora Crippen based on a scar she was said to have. Dr. Crippen was hanged, always proclaiming his innocence. In this study, genealogical research was used to locate maternal relatives of Cora Crippen, and their mitochondrial haplotypes were determined. Next, one of the pathology slides of the scar was obtained, DNA was isolated, and the haplotype was determined. That process was then repeated. Finally, both DNA isolates were assayed for repetitive elements on autosomes and repetitive elements specific to the Y chromosome. Based on the genealogical and mitochondrial DNA research, the tissue on the pathology slide used to convict Dr. Crippen was not that of Cora Crippen. Moreover, that tissue was male in origin.     

Value of DNA evidence in detecting crime

DNA material is now collected routinely from crime scenes for a wide range of offences and the timely processing of the DNA is seen as key to its success in investigating and detecting crime. An analysis of DNA material recovered from the volume crime offences of residential burglary, commercial burglary, and theft of motor vehicle in Northamptonshire, U.K., in 2004 has enabled the DNA to be categorized into seven sources. Further analysis using a logistical regression has revealed a number of predictors, other than timeliness, that greatly influence whether the DNA material recovered from a crime scene enables the crime to be detected. The results indicate that a number of these predictors are of statistical significance and may be just as relevant in determining whether DNA successfully detects the crime as the timeliness of the processing of the DNA material. The most significant predictor was found to be investigating officer accreditation with location, quantity, and type of DNA material at the crime scene also being relevant. Accreditation of the Crime Scene Examiner recovering the DNA material was found not to be significant. Consideration is given to where further emphasis is needed by the U.K. police service to maximize the opportunities to detect volume crime with DNA.     

Root Morphology and Anatomical Patterns in Forensic Dental Identification: A Comparison of Computer‐Aided Identification with Traditional Forensic Dental Identification*

Abstract: An online forensic dental identification exercise was conducted involving 24 antemortem–postmortem (AM–PM) dental radiograph pairs from actual forensic identification cases. Images had been digitally cropped to remove coronal tooth structure and dental restorations. Volunteer forensic odontologists were passively recruited to compare the AM–PM dental radiographs online and conclude identification status using the guidelines for identification from the American Board of Forensic Odontology. The mean accuracy rate for identification was 86.0% (standard deviation 9.2%). The same radiograph pairs were compared using a digital imaging software algorithm, which generated a normalized coefficient of similarity for each pair. Twenty of the radiograph pairs generated a mean accuracy of 85.0%. Four of the pairs could not be used to generate a coefficient of similarity. Receiver operator curve and area under the curve statistical analysis confirmed good discrimination abilities of both methods (online exercise = 0.978; UT‐ID index = 0.923) and Spearman’s rank correlation coefficient analysis (0.683) indicated good correlation between the results of both methods. Computer‐aided dental identification allows for an objective comparison of AM–PM radiographs and can be a useful tool to support a forensic dental identification conclusion.     

动物DNA分析在法庭科学中的应用

近年来,在法庭科学领域中,遇到越来越多的非人类DNA分型的问题,特别是来源于动物本身或者是动物的分泌物。作为证据,通过对犯罪现场非人类DNA的分型,不但可以知道在何地对何人或何物实施犯罪,而且,如果犯罪的实施方是动物,也可以知道其来自哪里。目前,在法医学领域,有关动物DNA分析方法的标准较少。根据国际法医遗传学会最新的研究成果,综述动物DNA在法庭科学中的应用现状和相关建议。     

Accuracy Rates of Sex Estimation by Forensic Anthropologists through Comparison with DNA Typing Results in Forensic Casework

A common task in forensic anthropology involves the estimation of the biological sex of a decedent by exploiting the sexual dimorphism between males and females. Estimation methods are often based on analysis of skeletal collections of known sex and most include a research‐based accuracy rate. However, the accuracy rates of sex estimation methods in actual forensic casework have rarely been studied. This article uses sex determinations based on DNA results from 360 forensic cases to develop accuracy rates for sex estimations conducted by forensic anthropologists. The overall rate of correct sex estimation from these cases is 94.7% with increasing accuracy rates as more skeletal material is available for analysis and as the education level and certification of the examiner increases. Nine of 19 incorrect assessments resulted from cases in which one skeletal element was available, suggesting that the use of an “undetermined” result may be more appropriate for these cases.     

Recovery of Trace DNA on Clothing: A Comparison of Mini‐tape Lifting and Three Other Forensic Evidence Collection Techniques

Trace DNA is often found in forensic science investigations. Experience has shown that it is difficult to retrieve a DNA profile when trace DNA is collected from clothing. The aim of this study was to compare four different DNA collection techniques on six different types of clothing in order to determine the best trace DNA recovery method. The classical stain recovery technique using a wet cotton swab was tested against dry swabbing, scraping and a new method, referred to as the mini‐tape lifting technique. Physical contact was simulated with three different “perpetrators” on 18 machine‐washed garments. DNA was collected with the four different DNA recovery methods and subjected to standard PCR‐based DNA profiling. The comparison of STR results showed best results for the mini‐tape lifting and scraping methods independent of the type of clothing. The new mini‐tape lifting technique proved to be an easy and reliable DNA collection method for textiles.     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

DNA分子标记技术在法医植物学中的应用

法医植物学是一门研究与法律事件相关的植物证据的科学。植物物证的DNA分子标记技术是近年来法医植物学研究的主要方向。本文系统地综述了目前应用于法医植物学中的各种DNA分子标记技术，对这些方法的实际应用案例以及可能应用方向进行列举，最后总结出这些分子生物学技术相较于传统植物形态分类方法的优点。     

Sexual Dimorphism of the Humerus in Contemporary Cretans—A Population‐Specific Study and a Review of the Literature*

Abstract: Sex determination is the first essential step for positive identification when a decomposed body is recovered. Taking into consideration the population aspect of sexual dimorphism of the skeleton, the present study aimed to create a sex identification technique using osteometric standards, derived from a contemporary Cretan population. A total of 168 left humeri were measured according to standard osteometric techniques. The differences between the means in males and females were significant (p < 0.0005). About 92.3% of cases were correctly classified when all measurements were applied jointly. Stepwise procedure produced an accuracy rate of 92.9%. The most effective single dimension was vertical head diameter (89.9%). The current study provides standards for a population that has not been represented so far in the existing databases. It demonstrates that the humerus is an effective bone for the estimation of sex because even in a fragmentary state it can give high classification accuracy.     

The Potential of Cosmetic Applicators as a Source of DNA for Forensic Analysis

Personal products, such as toothbrushes, have been used as both known reference and evidentiary samples for forensic DNA analysis. This study examined the viability of a broad selection of cosmetic applicators for use as targets for human DNA extraction and short tandem repeat (STR) analysis using standard polymerase chain reaction (PCR) conditions. Applicator types included eyeliner smudgers, pencils and crayons, eye shadow sponges, mascara wands, concealer wands, face makeup sponges, pads and brushes, lipsticks and balms, and lip gloss wands. The quantity and quality of DNA extracted from each type of applicator were examined by assessing the number of loci successfully amplified and the peak balance of the heterozygous alleles in each full STR profile. While degraded DNA, stochastic amplification, and PCR inhibition were observed for some items, full STR profiles were developed for 14 of 76 applicators. The face makeup sponge applicators yielded the highest proportional number of full STR profiles (4/7).     

Chemical Enhancement Techniques of Bloodstain Patterns and DNA Recovery After Fire Exposure*

Abstract: It is common in forensic casework to encounter situations where the suspect has set a fire to cover up or destroy possible evidence. While bloodstain pattern interpretation, chemical enhancement of blood, and recovery of deoxyribonucleic acid (DNA) from bloodstains is well documented in the literature, very little information is known about the effects of heat or fire on these types of examinations. In this study, a variety of known types of bloodstain patterns were created in a four‐room structure containing typical household objects and furnishings. The structure was allowed to burn to flashover and then it was extinguished by firefighters using water. Once the structure cooled over night, the interior was examined using a bright light. The bloodstains were evaluated to see if the heat or fire had caused any changes to the patterns that would inhibit interpretation. Bloodstain patterns remained visible and intact inside the structure and on furnishings unless the surface that held the blood was totally burned away. Additionally, a variety of chemical techniques were utilized to better visualize the patterns and determine the possible presence of blood after the fire. The soot from the fire formed a physical barrier that initially interfered with chemical enhancement of blood. However, when the soot was removed using water or alcohol, the chemicals used, fluorescein, luminol, Bluestar^®, and Hemastix^®, performed adequately in most of the tests. Prior to DNA testing, the combined phenolphthalein/tetramethyl benzidine presumptive test for the presence of blood was conducted in the laboratory on samples recovered from the structure in an effort to assess the effectiveness of using this type of testing as a screening tool. Test results demonstrated that reliance on obtaining a positive presumptive result for blood before proceeding with DNA testing could result in the failure to obtain useful typing results. Finally, two DNA recovery methods (swabbing the stain plus cutting or scraping the stain) were attempted to evaluate their performance in recovering samples in an arson investigation. Recovery of DNA was more successful in some instances with the swabbing method, and in other instances with the cutting/scraping method. Therefore, it is recommended that both methods be used. For the most part, the recovered DNA seemed to be unaffected by the heat, until the temperature was 800°C or greater. At this temperature, no DNA profiles were obtained.     

Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA Analysis,

Determining appropriate analytical thresholds (ATs) for forensic DNA analysis is critical to maximize allele detection. In this study, six methods to determine ATs for forensic DNA purposes were examined and compared. Four of the methods rely on analysis of the baseline noise of a number of negatives, while two utilize the relationship between relative fluorescence unit signal and DNA input in the polymerase chain reaction (PCR) derived from a dilution series ranging from 1 to 0.06 ng. Results showed that when a substantial mass of DNA (i.e., >1 ng) was amplified, the baseline noise increased, suggesting the application of an AT derived from negatives should only be applied to samples with low levels of DNA. Further, the number and intensity of these noise peaks increased with increasing injection times, indicating that to maximize the ability to detect alleles, ATs should be validated for each post‐PCR procedure employed.