Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.     

Whole-genome sequencing of degraded DNA for investigative genetic genealogy

Whole genome sequencing has opened the doors to Investigative genetic genealogy (IGG) analysis of challenging forensic samples that are not suitable for microarray genotyping. These samples still do not typically achieve high enough coverage for direct genotype calling, therefore a pipeline for imputation from low coverage sequencing data was evaluated using data from the 1000 Genomes Project. This pipeline generated results suitable for IGG down to 0.25X coverage. Additionally, forensic samples from a variety of tissue types and input amounts were sequenced and successfully uploaded to genetic genealogy databases after imputation.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.     

Analysis of microsatellite polymorphism in red deer, roe deer, and fallow deer -- possible employment in forensic applications

DNA microsatellites play a major role in population genetics, linkage mapping, and parentage studies of mammals. In addition, they may be used for forensic purposes, if an individual identification of a specific animal is necessary. Therefore, we tested a variety of microsatellite polymorphism derived from reindeer (Rangifer tarandus) by PCR and sequencing analysis for use in red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama). Twelve of these microsatellites were selected for further analysis. In all these microsatellite polymorphism short tandem repeats could be detected for one or all three species as shown by sequencing analysis. In red deer, more than two alleles were found in eight microsatellites, in roe deer more than two alleles could be demonstrated in seven microsatellites, whereas in fallow deer more than two alleles were found in only two microsatellite polymorphism. A comparison of sequences of PCR products from the three deer species with the sequences of reindeer revealed several differences between the four species. In six microsatellites -- selected because or their reliability in PCR and because of their polymorphic character -- we established a sequenced allelic ladder and give population data of all three species from 82 deer of the Northeast region of Germany (Vorpommern). Our results show the possibility to use microsatellite polymorphism in the identification of deer in forensic applications like poaching.     

Use of solid-phase double-antibody radioimmunoassay to identify species from small skeletal fragments

Protein radioimmunoassay (pRIA) offers the potential to identify species in small skeletal fragments submitted as forensic evidence. The technique consists of protein extraction followed by a solid-phase double-antibody radioimmunoassay using controls of antisera (raised in rabbits) and radioactive (iodine-125) antibody of rabbit gamma globulin (produced in donkeys). Species determination results from evaluation of radioactivity uptake. To demonstrate the potential of this technique, six known bone samples (three human and three nonhuman, including one from a deer [Odocoileus virginianus]) were submitted for blind analysis. pRIA correctly distinguished the human from the nonhuman samples. Using 200 mg or less of each sample, species of the deer specimen was identified correctly, given the choices of cow, deer, dog, goat, and pig.     

Content based information retrieval in forensic image databases

This paper gives an overview of the various available image databases and ways of searching these databases on image contents. The developments in research groups of searching in image databases is evaluated and compared with the forensic databases that exist. Forensic image databases of fingerprints, faces, shoeprints, handwriting, cartridge cases, drugs tablets, and tool marks are described. The developments in these fields appear to be valuable for forensic databases, especially that of the framework in MPEG-7, where the searching in image databases is standardized. In the future, the combination of the databases (also DNA-databases) and possibilities to combine these can result in stronger forensic evidence.     

Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.     

The use of isoelectric focusing to identify rhinoceros keratins.

Keratins represent the principal structural proteins of hair. They are also found in horn, nail, claw, hoof, and feather. Hair and nail samples from human and canine sources and hair samples from mule deer, white tail deer, cat, moose, elk, antelope, caribou, raccoon, and goat were studied. Parrot and goose feathers were also analyzed. Keratins are polymorphic, and species differences are known to exist. Proteinaceous extracts of deer and antelope antlers and bovine and rhinoceros horn were prepared by solubilizing 10 mg of horn sample in 200 microL of a solution containing 12M urea, 74mM Trizma base, and 78mM dithiothreitol (DTT). Extraction took place over a 48-h period. A 25-microL aliquot of extract was removed and incubated with 5 microL of 0.1 M DTT for 10 min at 25 degrees C. Keratins were then separated by isoelectric focusing (IEF) on 5.2% polyacrylamide gels for 3 h and visualized using silver staining. At least 20 bands could be observed for each species studied. However, band patterns differed in the position of each band, in the number of bands, and in band coloration resulting from the silver staining process. Horn from two species of rhinoceros was examined. For both specimens, most bands occurred in the pH range of 4 to 5. Although similar patterns for both species were observed, they differed sufficiently to differentiate one from the other. As might be expected, the closer two species are related phylogenetically, the greater the similarity in the IEF pattern produced from their solubilized keratin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recommendations for consistent treatment of length variants in the human mitochondrial DNA control region

Human mitochondrial DNA (mtDNA) analysis is a valuable forensic tool, useful in cases where the amount of extracted DNA is low or highly degraded. Population databases are used to determine the relative rarity of a particular profile obtained in a forensic case. Rather than full DNA sequence information, sequence profiles are compared to a reference sequence, and the differences from the reference are recorded in forensic databases. A standard method is proposed for characterizing length variants, and examples are described using actual human control region mtDNA profiles. Consistency in alignment and nomenclature avoids inadvertently describing two sequences as different when in fact they are the same.     

Identifying the scene of a crime through pollen analysis

The forensic analysis of pollen involves the comparison of crime scene and reference pollen samples. Successful matches are frequently used to solve time- or location-related crimes. Despite its prospects in criminal investigation, forensic palynology is still underused in casework due to inherent shortcomings such as its limited evidential weighting, scarcity of skilled palynologists dedicated to forensic casework and the laborious nature of analytical procedures. To address these challenges, the current state-of-the-art in forensic palynology is transiting from the traditional light microscopic methods that dominated the early days of palynology to more contemporary approaches like Raman spectroscopy, stable isotope analysis and DNA metabarcoding. The major challenges of these methods, however, include a lack of optimisation to forensic expectations and the unavailability of robust databases to permit accurate data interpretation, and quests to resolve these problems constitute the theme of current research. While reiterating the usefulness of pollen analysis in criminal investigation, this report recommends orthogonal testing as a way of improving the evidential weighting of forensic palynology.     

Retention of Offender DNA Samples Necessary to Ensure and Monitor Quality of Forensic DNA Efforts: Appropriate Safeguards Exist to Protect the DNA Samples from Misuse

Retention of offender DNA samples serves an important quality assurance role for forensic DNA laboratories. Consistent with the principles of confidentiality underlying the establishment of the state and national DNA databases, safeguards are in place to protect the DNA samples from unauthorized use.     

Forensic STR analysis reveals DNA contamination previously undetected during clinical analysis of chronically inflamed tissues

While investigating the potential for genetic instability in chronic inflammatory disease, using ulcerative colitis (UC) as a model, we analyzed microsatellite DNA of both pre- and post-surgical affected and histologically normal tissues. These samples were also characterized using the forensic Identifiler^® Multiplex System from ABI. Apparent instability was found in the majority of patients using the clinical panel. This panel assumed all samples were single source, whereas the forensic panel revealed that 57% of samples tested with Identifiler^® were mixtures of more than one contributor. It is likely that DNA contamination occurred during routine histological processing. This contamination could lead to erroneous assessments of instability. Microsatellite analysis is used in tumor characterization and therapeutic determinations. Incorrect determinations could affect patient care. Given the sensitivity and widespread use of molecular tests on biopsies and preserved post-surgical tissues, we recommend that an STR multiplex used for forensic individualization be used prior to diagnostic tests to ensure the sample is from a single source.     

PCR扩增SON基因3’非编码区进行种属鉴定

根据人 DNA的 SON基因碱基序列选择性设计一对特异性引物 ,并对人和猕猴、阿拉伯狒狒、猪、牛、羊、马、驴、骡、狗、猫、兔、大白鼠、小白鼠、豚鼠等 14种哺乳类动物染色体 DNA的 SON基因 3’非编码区中的种属特异性区域进行 PCR扩增 ,扩增产物经 SSCP分离银染色技术检测 ,观察到人和 14种哺乳类动物的 SSCP图谱有明显不同。本方法可以对人和上述 14种哺乳类动物进行种属鉴定 ,适合于法医学种属鉴定的应用     

“It all happened so slowly” – On controlling function creep in forensic DNA databases

Forensic DNA databases are implemented worldwide and used increasingly. Part of this increasing usage is arguably a matter of function creep. Function creep refers to changes in, and especially additions to, the use of a technology. In this article we explore the notion of function creep as we discuss why and how it has taken place on forensic DNA databases. We also consider what future function creep it is possible to envisage. As even security enhancing technologies may contribute to insecurities, what safeguards should be in place to render function creep governable? We use the Norwegian DNA database, expanded considerably as recently as September 2008, as our primary case for discussion. Additionally we use examples from the English and Welsh DNA database which, considered world leading, may be an indication of where other DNA databases are heading. The article isn't data-driven but draws on a wide spectrum of data: governmental documents, public and Parliamentary debates, and interviews.     

The use of cadaver dogs in locating scattered, scavenged human remains: preliminary field test results

Specially trained air scent detection canines (Canis familiaris) are commonly used by law enforcement to detect narcotics, explosives or contraband, and by fire investigators to detect the presence of accelerants. Dogs are also used by police, military, and civilian groups to locate lost or missing persons, as well as victims of natural or mass disasters. A further subspecialty is "cadaver" searching, or the use of canines to locate buried or concealed human remains. Recent forensic investigations in central Alberta demonstrated that the use of cadaver dogs could be expanded to include locating partial, scattered human remains dispersed by repeated animal scavenging. Eight dog-and-handler teams participated in a two-month training program using human and animal remains in various stages of decay as scent sources. Ten blind field tests were then conducted which simulated actual search conditions. Recovery rates ranged between 57% and 100%, indicating that properly trained cadaver dogs can make significant contributions in the location and recovery of scattered human remains.     

Evidence in support of self-declaration as a sampling method for the formation of sub-population DNA databases

Well constructed sub-population databases are fundamental to the application of DNA-based forensic statistics. The size of such databases can affect the ability to examine adequately statistical or population genetic features, and the integrity of both the DNA profile and associated ethnicity information is also of importance. Use of short tandem repeat (STR) DNA profiling technology and the thoughtful construction of the governing legislation has seen large databases of DNA profiles collated for the four major sub-populations of New Zealand. Examination of the data illustrates the suitability of self-declaration as a means of categorizing samples on the basis of ethnicity.     

A comprehensive study into false positive rates for ‘other’ biological samples using common presumptive testing methods

The identification of biological fluids or materials in forensic samples is a key requirement in forensic science that relies on chemical and biological based tests, most of which exhibit false positivity. When reporting results from such tests, Forensic Scientists use words such as probable, possible, and likely, without always being able to provide robust support for these conclusions. In collating information about false positive rates for a number of these tests, we found limited research into the cross reactions observed from ‘other’ biological samples in commonly encountered case sample stains. By ‘other’ we mean biological fluids or materials that are not the primary target of the presumptive test being used. Here we carry out a specificity study to fill gaps in the literature for a number of the presumptive chemical, biological and immunochromatographic tests used to presumptively screen for blood, semen and saliva. The tests selected for this study are the widely used tests: Luminol, TMB/Combur³ Test® E, Kastle-Meyer (KM), RSID™ - Blood, ABAcard® HemaTrace®, Acid Phosphatase (AP), ABAcard® p30, RSID™ - Semen, Phadebas® ‘Tube’ Test, Phadebas® ‘Press’ Test, and RSID™ - Saliva tests. Specificity for each of these was tested in known samples, from volunteers, of blood, semen, saliva, urine, sweat, vaginal material, faeces and breast milk, and then false positive rates were determined.     

Evaluation of prostate-specific antigen (PSA) membrane test assays for the forensic identification of seminal fluid.

Prostate specific antigen (PSA, also known as p30), a glycoprotein produced by the prostatic gland and secreted into seminal plasma, is a marker used for demonstrating the presence of seminal fluid. Methods for the detection of PSA include Ouchterlony double diffusion, crossover electrophoresis, rocket immuno-electrophoresis, radial immunodiffusion, and ELISA. The extremely sensitive ELISA technique can detect PSA in concentrations as low as approximately 4 ng/mL. However, all these techniques are cumbersome and time consuming to perform in forensic laboratories, especially when only a few samples per week are processed. Various membrane tests are currently used in clinical settings to screen a patient's serum for the presence of PSA at levels greater than 4 ng/mL. In this study we evaluated three immunochromatographic PSA membrane tests by analyzing semen stains stored at room temperature for up to 30 years, post-coital vaginal swabs taken at different time after intercourse, semen-free vaginal swabs, and various female and male body fluids, including urine. The data demonstrate that PSA membrane test assays offer the same sensitivity as ELISA-based tests and provide a rapid approach for the forensic identification of seminal fluid. Furthermore, when the supernatant from a DNA extraction is used for the assay, there is essentially no DNA consumption for determining the presence of PSA in a forensic sample.