Article

Forensic Evidence Based on mtDNA from Dog and Wolf Hairs

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Abstract

In six forensic cases involving murder, bank robbery, theft and poaching, evidence material comprising shed hairs supposedly originating from dogs or wolves was analyzed by mitochondrial (mt) DNA sequencing. A 79 bp segment of the control region was amplified, sequenced, and compared with an established database of the domestic dog and wolf populations. In three murder cases exclusions of all eight suspects could be made. Furthermore, two of the murders could be linked to each other by a rare sequence variant, and the breed of the dog was indicated. In a theft case and a bank robbery a link could be established between the evidence material and the suspects. In a case of suspected wolf poaching, it could be established that the evidential material was of dog rather than wolf origin. We conclude that single hairs from common pets are suitable for DNA analysis and that the described method has proved to be a valuable tool for forensic investigations.

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... Hair is a characteristic for one vertebrate class in particular, Mammalia, although hair cells are also present in other vertebrates, for example in lateral line organs of fishes and amphibians where they detect movement relative to the environment (Campbell, 1996). The hairs that are found at most crime scenes are mostly shed hairs of human origin, or from pets (Menotti-Raymond et al, 1997;Savolainen et al, 1999). In Sweden, for example, ~40 % of all households have either cats or dogs (Manimalis, 2005). ...
... Analysis of non-human DNA for forensics can be a valuable aid not only in crime investigations involving a human perpetrator and a human victim but also in a wide range of other cases. Applications of non-human DNA analysis include investigations of illegal trade in endangered species or poaching (Guglich et al, 1994;Miller et al, 1995;Savolainen et al, 1999;Singh et al, 2004;Wetton et al, 2004), animal-attacks (Brauner et al, 2001;Eichmann et al, 2004), non-declared, wrongly declared or forbidden animal products in human food and animal feed (Meyer et al, 1996;Dalmasso et al, 2004), traffic accidents (Schneider et al, 1999); fishing competition fraud (Primmer et al, 2000) and medical negligence such as HIV transmission (Ou et al, 1992). There are many cases where plant or animal DNA has been found in association with murder. ...
... ago; Ingman et al, 2000), giving much less time for variation to evolve among domestic dogs than among humans. Very few forensic cases involving analysis of domestic dog mtDNA have been published in scientific journals (Savolainen et al, 1999;Schneider et al; although there are other cases where the method was used (for example, see www.questgen.biz/CV.htm). Six cases from Sweden (Savolainen et al, 1999) are addressed below to illustrate different situations where of mtDNA analysis of shed hairs from domestic dogs can be useful for forensic casework: ...
... Twenty-one variable and informative sites were observed in this study, with 19 of the sites shared by both breeds. All of the shared sites were highly informative (15,526, 15,595, 15,612, 15,620, 15,627, 15,632, 15,639, 15,643, 15,652, 15,800, 15,815, 15,912, 15,955, 16,003, 16,025, 16,083, 16,128, 16,431, 16,439). The differences included one rare site (16,032) for Labrador Retrievers and a common dog SNP (16,672) observed in Golden Retrievers only. ...
... Twenty-one variable and informative sites were observed in this study, with 19 of the sites shared by both breeds. All of the shared sites were highly informative (15,526, 15,595, 15,612, 15,620, 15,627, 15,632, 15,639, 15,643, 15,652, 15,800, 15,815, 15,912, 15,955, 16,003, 16,025, 16,083, 16,128, 16,431, 16,439). The differences included one rare site (16,032) for Labrador Retrievers and a common dog SNP (16,672) observed in Golden Retrievers only. ...
... Twenty-one variable and informative sites were observed in this study, with 19 of the sites shared by both breeds. All of the shared sites were highly informative (15,526, 15,595, 15,612, 15,620, 15,627, 15,632, 15,639, 15,643, 15,652, 15,800, 15,815, 15,912, 15,955, 16,003, 16,025, 16,083, 16,128, 16,431, 16,439). The differences included one rare site (16,032) for Labrador Retrievers and a common dog SNP (16,672) observed in Golden Retrievers only. ...
Article
The mitochondrial DNA (mtDNA) control regions of 125 domestic dogs (Canis familiaris) encompassing 43 breeds, as well as one coyote and two wolves were sequenced and subsequently examined for sequence variation in an effort to construct a reference dog mtDNA data set for forensic analysis. Forty informative variable sites were identified that described 45 haplotypes, 29 of which were observed only once. Substantial variation was found both within and among breeds in the mtDNA derived from tissue, indicating that analysis of the mtDNA derived from dog hairs could be a valuable, discriminating piece of evidence in forensic investigations. The dog data set single nucleotide polymorphisms (SNPs) ranged from having one to six changes on a phylogenetic tree. On average, there were 1.9 character changes for each variable position on the tree. The most variable sites (with four or more changes each, listed from the most changes to the fewest) observed were 15,639 (L=6), 16,672 (L=5), 15,955 (L=4), 15,627 (L=3), 16,431 (L=3), and 16,439 (L=3). These sites were consistent with other reports on variable positions in the dog mtDNA genome. A total of 26 SNPs were chosen to best identify all major clusters in the domestic dog data set. The descriptive analyses revealed that this data set is similar to other published canine data sets and further demonstrates that this domestic dog data set is a useful resource for forensic applications. This reference data set has been compiled and validated against the published dog genetic literature with an aim to aid forensic investigations that seek to incorporate mtDNA sequences and SNPs from trace evidence such as dog hair.
... The 148-bp segment of the hypervariable region 1 (HV1) was amplified using the primers D5 and D8 [1]. In the cases where PCR product was not detectable by agarose gel electrophoresis, seminested PCR was carried out, using the primer D8 and the primer D7 [1]. ...
... The 148-bp segment of the hypervariable region 1 (HV1) was amplified using the primers D5 and D8 [1]. In the cases where PCR product was not detectable by agarose gel electrophoresis, seminested PCR was carried out, using the primer D8 and the primer D7 [1]. The size of the product of the second PCR amplification was reduced to 125 bp. ...
... The primer D8 was designed to carry M13 -21 sequence at the 5V-end to enable direct sequencing of PCR products. The PCR conditions were in accordance with the literature [1]. PCR products were fractionated by agarose gel electrophoresis. ...
Article
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Dog's hairs and possible saliva stains were recovered from the coat of a dead woman, possibly attacked by a dog(s). The reference hairs and saliva samples were collected from different dogs living in the neighbourhood. Mitochondrial and nuclear DNA analysis were used to analyze the crime scene and reference samples. Despite many samples analyzed and a lot of DNA data obtained, it was not possible to determine which dog's hair and saliva were on the murdered woman's coat, as all reference dogs/samples had been excluded.
... A farkasok és kutyák első, filogenetikai irányú analízise már csaknem 25 évvel ezelőtt kezdődött a mitokondriális KR vizsgálatával [32], amit gyorsan követtek további genetikai tesztelések filogeográfiai, hibridizáció és igazságügyi azonosítás, valamint a genetikai diverzitás felmérése céljából [17,[46][47][48][49][50][51][52]. Bár az mtDNS csak anyai ágon öröklődik, így egyedüli alkalmazása nem ad teljes képet hibridizációs kérdésekben, a KR vizsgálata mégis hasznos módszernek bizonyult későbbi kutatásokban [6,[53][54][55]. ...
... Az ilyen igazságügyi esetek hátterének felderítéséhez általában különböző típusú molekuláris genetikai markereket, ill. ezek analízisének ötvözetét használják [2,47,[64][65][66]. Munkánk során mi is megkezdtük azon markerek tesztelését, amelyek a kitűzött céljainknak leginkább megfelnek, és az összesített eredményeink alapján többnyire sikeresnek bizonyultak a bi-és uniparentális módszerekkel végzett genetikai kutatásaink. ...
Article
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ÖSSZEFOGLALÁS A szerzők a kutyák és farkasok különböző genetikai markerekkel történő elkülönítésének lehetőségeit vizsgálják és bemutatják ennek fontosságát az igazságügyi alkalmazás területén. A rendelkezésre álló farkas-és kutyaeredetű mintákból meghatározott mitokondriális kontrollrégió haplotípusok és a 14 vizsgált mikro-szatellita-alléleloszlás adatai alapján különbség látható a farkas-és kutyaminták között. Az eddigi hazai eredmények is alátámasztják annak lehetőségét, hogy különböző genetikai markerek párhuzamos vizsgálatával − amelyek megfelelnek az igazságügyi célú alkalmazás kritériumainak −, kellő valószínűséggel alátámasztható egy kérdéses eredetű minta alfajszintű besorolása. SUMMARY Background: After several decades of absence, the grey wolf (Canis lupus) has started recolonizing its former territories in Hungary at the beginning of the 21 st century. Due to the intense presence of mankind, wolves are forced to share great areas of land with humans, which potentially leads to several conflicts. From the wolves' perspective, it means the decimation of domestic livestock. As far as humans are concerned, these conflicts may manifest in the illegal hunting of wolves and trading with their products. When facing such case, it should be examined whether the perpetrator/victim is a wolf or a dog. Objective: The aim of our study was to test genetic methods which can be used for forensic application as well to distinguish between wolves, dogs, or their hybrids. Materials and Methods: Altogether 22 samples (hair, skin, faeces, saliva, and purified DNA) from wolves and wolf-dog hybrids were collected. For the comparative canine database DNA samples from Hungarian dog populations were used. After DNA isolation, the mitochondrial hypervariable region I (HVI) and 14 autoso-mal microsatellite markers were amplified by PCR (Polymerase Chain Reaction). Mitochondrial haplotypes determined by sequencing were grouped using PopART. Genetic profiles based on the detected microsatellite alleles were analysed using Structure 2.3.4 and were grouped based on a Bayesian approach. Results and Discussion: The mitochondrial control region (HVI) haplotypes were successfully determined from the examined samples; these sequences were uploaded to the GenBank database. We did not find similar point mutation patterns between wolves and dogs. However, difference between wolf and dog groups was shown based on the detected microsatellite allele distribution, to make the results even more reliable further markers and more wolf samples should be involved. Overall, our preliminary results support that simultaneous application of large number of genetic markers meeting the standards of forensic application criteria-, could be adequate to determine the precise taxonomic origin of questionable samples.
... Just like the human HV1, the canine HV1 is highly polymorphic and is of keen forensic interest because it can also be successfully amplified and typed from limited or severely degraded DNA (5)(6)(7). Sufficient mtDNA can be reliably typed from a single dog hair shaft (1,5,(8)(9)(10)(11). ...
... However, in the database of 64 haplotypes, a 4% increase in exclusion capacity, i.e., from 0.89 to 0.93, was obtained despite the increase in sample size from only 36 dogs to 237-almost a sevenfold increase in sampling size. A study by Savolainen et al. (7,9) yielded comparable results from their analysis of 52 of the most common dog breeds in Sweden. With a sampling of 102 Swedish dogs, they were able to calculate an exclusion capacity of 0.88 by analyzing a 257-bp segment of the control region containing HV1. ...
Article
  The 608-bp hypervariable region 1 (HV1) sequences from 36 local dogs were analyzed to characterize the population genetic structure of canid mitochondrial DNA (mtDNA). Sixteen haplotypes were identified. A 417-bp segment of this sequence was compared with GenBank sequences from a geographically representative sample of 201 dogs, two coyotes, and two wolves. Sixty-six haplotypes were identified including 62 found only in domestic dogs. Fourteen of these correspond to the 16 local haplotypes and were among the most frequent haplotypes. The local sample was judged to be representative of the much broader geographic sample. No correlation was observed between local haplotypes and the owner’s characterization of dog breed. A 60-bp variation “hotspot” within the canid HV1 was identified as a potentially valuable molecular tool, particularly for assaying limited or degraded DNA samples.
... Biological materials from pure breed or mixed breed dogs have been used to establish important links in human criminal cases such as traffic accidents [2], murders [State of California vs. David Westerfield, 2002], bank robberies [3], and dog attack cases where there are human [4] or nonhuman victims [5]. In the US alone, there are estimated to be between 3.5 and 4.7 million dog bite injuries to humans annually [6]. ...
... Concordant with the fixation indices, the high average numbers of inter-and intrapopulation pairwise Fst differences within the combined, pure breed, and mixed breed datasets reveal that there is substantial genetic differentiation among the different regional populations ( Table 4). Estimates of the PE for each of the combined, pure breed, and mixed breed datasets are presented in Table 5 and are consistent with the previously reported range of the 0.86 -0.95 [7]. 2 The asterisk ( * ) denotes the pairwise Fst comparison that is significantly different from random expectation at the 0.05 level 3 The values above the diagonal equal the average number of pairwise differences between regional populations. The values on the diagonal equal the average number of pairwise differences within regional populations, and the values below the diagonal equal a corrected average pairwise difference. ...
Article
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No geographic differences in mitochondrial DNA (mtDNA) distribution among United States (US) domestic dog populations have been detected to date. To test the hypothesis that regional differences exist, a 608 bp sequence of the canid mtDNA hypervariable region 1 (HV1) from 220 mixed breed animals from the Western, Northeastern, Midwestern, and Southern US were combined with 429 published mixed and pure breed dog HV1 sequences to form a substantial geographically representative dataset. With an increased sample size of regionally representative sequences, geographic substructure among regional populations was shown to be statistically significant using the modified Fisher's exact test and pairwise Fst. The results of the AMOVA showed that 91% of the variation is present within the regional dog populations. Based on these analyses, the significance of regional canine HV1 haplotype distributions and frequencies demonstrate further the value of regional and mixed breed canine mtDNA in forensic investigations in the US.
... The popularity of domestic dogs (Canis lupus familiaris) combined with their close proximity to humans results in the frequent recovery of canine biological evidence, particularly hair, from crime scenes [1][2][3]. Most hair shafts recovered from crime scenes are shed hair-not pulled hair-and do not have the growing root bulb or attached soft tissue necessary for nuclear DNA profiling [4]. ...
... In contrast, the high copy number of circular maternally inherited mitochondrial DNA (mtDNA) genomes present in each hair shaft permits mtDNA haplotyping of a single dog hair [5,6]. Forensic evidence based on mtDNA from dog hairs has been used to include or exclude suspects in cases such as murder, theft, robbery, and suspected poaching of wolves [1,2,7]. However, the analysis of mtDNA in forensic science can be complicated by heteroplasmy-the co-existence of subpopulations of mtDNA genomes between or within individual cells or mitochondria [8]. ...
... Results on dog hair mtDNA analyses have been published previously. Savolainen and colleagues presented results on data obtained from single plucked hairs extracted using a robotic workstation [1] and on single shed hairs in casework examples [6]. In the same year, Schneider and colleagues also reported on the successful analysis of a single dog hair (presumably shed hair) found at a scene of a traffic accident [7] while a paper by Angleby and Savolainen from 2005 presented results on 2-5 plucked hairs [8]. ...
... Methods have been published for the extraction of DNA from dog hairs with roots [1,8,17,18] and without roots before with varying success rates [6,9,10]. In this paper, a straightforward protocol is presented for the automated extraction of single dog hairs resulting in complete and good quality mtDNA control region sequences. ...
... I British Colombia hvor det er rapportert fatale angrep av puma og bjørn blir alle dødsfall i utgangspunktet betraktet som kriminelle handlinger og det blir lagt spesiel l vekt på å sikre bevis på åstedet. Mul igheten for å bruke DNA-metoder til å identifisere identiteten til det ansvarlige dyret bør også undersøkes (Savolainen & Lundeberg 1999). ...
... The canine mtDNA sequence has been used to clarify the origin of domestic dogs ). The canine mtDNA sequence has been used to clarify the origin of domestic dogs ( (SAVOLAINEN et al., 2002;VILÀ et al., 1997;PANG et al., 2009SAVOLAINEN et al., 2002VILÀ et al., 1997;PANG et al., 2009), assess ), assess hybridization with wild species ( hybridization with wild species (VILÀ et al., 1997;ADAMS et VILÀ et al., 1997;ADAMS et al., 2003a;ADAMS et al., 2003b;al., 2003a;ADAMS et al., 2003b;CIUCCI et al., 2003;VILÀ et al., 2003;VERGINELLI et al., CIUCCI et al., 2003;VILÀ et al., 2003;VERGINELLI et al., 2005;SCHMUTZ et al., 2005 and forensic analysis ( ) and forensic analysis (SAVOLAINEN and LUNDEBERG, 1999;SAVOLAINEN and LUNDEBERG, 1999;WETTON et al., 2003WETTON et al., 2003. Several authors ( ). ...
... The DNA obtained from shed or plucked hairs is useful for genetic analysis in forensic investigations (Pascali et al., 1994;Allen et al., 1998;Menotti-Raymond et al., 1997;Savolainen and Lundeberg, 1999) and for questions of the population structure and molecular evolution of a variety of species (Vigilant et al., 1989;Morin et al., 1994;Garner and Ryder, 1996;Taberlet et al., 1997;Goossens et al., 1998;Lum et al., 1998;Gagneux et al., 1999). Many studies have examined only the more abundant mtDNA because nuclear DNA targets are more difficult to amplify, particularly from shed rather than plucked specimens (Goldberg and Ruvolo, 1997;Saltonstall et al., 1998). ...
Article
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Hair can be a valuable source of DNA for the noninvasive study of human and nonhuman populations. However, hairs contain extremely small quantities of DNA, making the method used to extract the DNA of paramount importance. This study compares the effectiveness of 4 different methods of DNA extraction from shed chimpanzee hair, as measured by the ability to amplify mtDNA targets using PCR. The most successful method is also the simplest, requiring only digestion of the root end in a buffer compatible with subsequent PCR without a prior purification or extraction step. Strategies to non-specifically preamplify the template are not successful with DNA from stored shed hairs.
... The identification of an individual pet or other animal may provide the critical piece of information in a criminal investigation and prosecution. With the majority of American households (55%) housing at least one cat or dog (73 million cats, 68 million dogs) (1), it is not unusual for animal specimens, particularly hair specimens, to be part of the physical evidence associated with a crime scene (2)(3)(4)(5)(6)(7). A study on the transfer and persistence of animal hair has demonstrated that it is almost impossible to enter a house where a domestic animal lives without becoming a ''carrier'' of its hair (8). ...
Article
A simple tandem repeat (STR) PCR-based typing system developed for the genetic individualization of domestic cat samples has been used to generate a population genetic database of domestic cat breeds. A panel of 10 tetranucleotide STR loci and a gender-identifying sequence tagged site (STS) were co-amplified in genomic DNA of 1043 individuals representing 38 cat breeds. The STR panel exhibits relatively high heterozygosity in cat breeds, with an average 10-locus heterozygosity of 0.71, which represents an average of 38 breed-specific heterozygosities for the 10-member panel. When the entire set of breed individuals was analyzed as a single population, a heterozygosity of 0.87 was observed. Heterozygosities obtained for the 10 loci range from 0.72 to 0.96. The power for genetic individualization of domestic cat samples of the multiplex is high, with a probability of match (p(m)) of 6.2E-14, using a conservative θ = 0.05.
... Since then, the assignment of non-human DNA samples to a particular individual has been routinely used [2]. Reported forensic casework involving individual identification of animals includes many types of investigation, ranging from poaching of a wild boar [3] to armed robbery [4]. Unlike taxonomic identification of evidence, which can be performed based on morphological analysis or through DNA sequencing of universal barcoding genes, individual identification requires genetic analyses of multiple, taxon-specific, polymorphic DNA markers such as short tandem repeat (STR) or single nucleotide polymorphism (SNP) loci [5]. ...
Article
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European roe deer (Capreolus capreolus L.) are the most common game species in Europe, hunted for meat and trophies. Forensic investigations involving roe deer poaching may often benefit from an individual identification method to link a suspect to a specific incident. The current paper presents a forensically validated DNA profiling system for European roe deer called “STRoe deer”. This DNA profiling system consists of 12 novel unlinked tetra-nucleotide short tandem repeat (STR) loci and two sexing markers, with an allelic ladder to facilitate accurate genotyping. Validation results using 513 European roe deer samples collected from a single population from the Swiss Plateau demonstrated successful amplification of all 14 loci with as little as 0.05 ng of European roe deer DNA. Species-specificity tests showed that other members of the Cervidae family exhibited partial profiles and non-specific peaks, whereas most members of the Bovidae family showed just non-specific cross-species amplification products. Three different methods to calculate match probabilities for randomly sampled European roe deer genotypes resulted in median match probabilities ranging from 1.4 × 10⁻¹³ to 2.5 × 10⁻⁵. These methods accounted for possible population structure, occurrence of null alleles and individual relatedness. Based on these results, we conclude that STRoe deer is a robust genotyping system that should prove a valuable tool for individual identification and sexing of European roe deer to support criminal investigations.
... DNA barcoding uses small segments of DNA to identify species and has been effectively used for species-level identification in many animal and plant groups (Hebert et al., 2003;CBOL Plant Working Group, 2009), invasive species control (Floyd et al., 2010), forensics (Savolainen and Lundeberg, 1999), and regulatory enforcement (Parveen et al., 2016). Although DNA barcoding is unlikely to replace the field identification of species, it is another tool for when morphological features are not available, whether due to disturbance (e.g., grazing or burning), for the analysis of fecal material (Goldberg et al., 2020), or for the verification of morphology-based identification. ...
Article
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Premise: The tallgrass prairies of North America are one of the most threatened ecosystems in the world, making efficient species identification essential for understanding and managing diversity. Here, we assess DNA barcoding with high-throughput sequencing as a method for rapid plant species identification. Methods: Using herbarium collections representing the tallgrass prairie flora of Oak Lake Field Station, South Dakota, USA, we amplified and examined four common nuclear and plastid barcode regions (ITS, matK, psbA-trnH, and rbcL), individually and in combination, to test their success in identifying samples to family, genus, and species levels using BLAST searches of three databases of varying size. Results: Concatenated barcodes increased performance, although none were significantly different than single-region barcodes. The plastid region psbA-trnH performed significantly more poorly than the others, while barcodes containing ITS performed best. Database size significantly affected identification success at all three taxonomic levels. Confident species-level identification ranged from 8-44% for the global database, 13-56% for the regional database, and 21-80% for the sampled species database, depending on the barcode used. Discussion: Barcoding was generally successful in identifying tallgrass prairie genera and families, but was of limited use in species-level identifications. Database size was an important factor in successful plant identification. We discuss future directions and considerations for improving the performance of DNA barcoding in tallgrass prairies.
... For this reason, coding regions of the mitochondrial genome have either no resolution between wolf and dog (12S and 16S ribosomal RNA genes) 25,26 , or detectable but poor resolution (Cyt b and COI genes) 27 . The control region (CR) holds a large number of polymorphic sites in the neutrally evolving hypervariable regions (HV) 28,29 , where some separation of wolf versus dog have been successful 30,31 . However, identification based on CR sequence may not be reliable unless additional nuclear markers are combined 32,33 especially when the reference sample size is not sufficient to represent total genetic variation 34 . ...
Article
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Wolf (Canis lupus) is a species included in appendices of CITES and is often encountered in cases of alleged poaching and trafficking of their products. When such crimes are suspected, those involved may attempt to evade legal action by claiming that the animals involved are domestic dogs (C. l. familiaris). To respond effectively to such claims, law enforcement agencies require reliable and robust methods to distinguish wolves from dogs. Reported molecular genetic methods are either unreliable (mitogenome sequence based), or operationally cumbersome and require much DNA (un-multiplexed microsatellites), or financially expensive (genome wide SNP genotyping). We report on the validation of a panel of 12 ancestral informative single nucleotide polymorphism (SNP) markers for discriminating wolves from dogs. A SNaPshot multiplex genotyping system was developed for the panel, and 97 Mongolian wolves (C. l. chanco) and 108 domestic dogs were used for validation. Results showed this panel had high genotyping success (0.991), reproducibility (1.00) and origin assignment accuracy (0.97 ± 0.05 for dogs and 1.00 ± 0.03 for wolves). Species-specificity testing suggested strong tolerance to DNA contamination across species, except for Canidae. The minimum DNA required for reliable genotyping was 6.25 pg/μl. The method and established gene frequency database are available to support identification of wolves and dogs by law enforcement agencies.
... Cycle sequencing of a part of the cytochrome b gene located on the mtDNA was performed in order to determine the animal species of the hairs found on the victim's corpse [1]. Furthermore a part of the hypervariable control region was analyzed from all the evidence samples originating from dogs [2]. The haplotypes were aligned with the haplotype of reference hairs from dogs found in the suspect's car. ...
Article
In January 2005 the dead body of a young female was found close to a highway in the south of Austria. Since the corpse had been set on fire using an accelerant, the victim has not been identified so far. Part of the sweater the woman was wearing was left and showed few short animal hairs. The investigations led the police to a young man who was already on remand due to a property offense. On the basis of morphological comparison, the hairs found on the sweater of the victim were suspected to derive either from an animal out of the group of minks and martens (Mustelidae) or from a dog (Canidae). Since hardly any of the hairs showed a root, mtDNA analysis had to be performed on the few hairs collected from the victim's sweater and on the hairs found in the suspect's car. The questions posed by the court were as follows: which species do the hairs on the sweater originate from, and secondly: do the hairs on the sweater and the hairs in the car belong to the same individual.
... Because of its high level of length heteroplasmy, this repeat region is mostly not considered in forensics (Fridez et al. 1999). Several publications illustrate forensic casework involving control region analysis of dog traces, such as Savolainen and Lundeberg (1999), Schneider et al. (1999), Branicki et al. (2002), Aaspõllu and Kelve (2003), Halverson and Basten (2005) and Scharnhorst and Kanthaswamy (2011). ...
Article
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The identification of dog hair through mtDNA analysis has become increasingly important in the last 15 years, as it can provide associative evidence connecting victims and suspects. The evidential value of an mtDNA match between dog hair and its potential donor is determined by the random match probability of the haplotype. This probability is based on the haplotype's population frequency estimate. Consequently, implementing a population study representative of the population relevant to the forensic case is vital to the correct evaluation of the evidence. This paper reviews numerous published dog mtDNA studies and shows that many of these studies vary widely in sampling strategies and data quality. Therefore, several features influencing the representativeness of a population sample are discussed. Moreover, recommendations are provided on how to set up a dog mtDNA population study and how to decide whether or not to include published data. This review emphasizes the need for improved dog mtDNA population data for forensic purposes, including targeting the entire mitochondrial genome. In particular, the creation of a publicly available database of qualitative dog mtDNA population studies would improve the genetic analysis of dog traces in forensic casework.
... In forensic science, much attention has been paid to the identification of human individuals [1,2]. However, the identification of non-human animal species is also important in many areas, for example, criminal investigations [3][4][5], wildlife forensics [6][7][8], forensic entomology [9][10][11], and food authentication [12,13]. In particular, the identification of non-human mammalian species is useful in criminal investigations. ...
Article
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Mammalian species identification is one of the important issues in forensic science. Determining the origins of non-human biological material found at crime scenes can increase the possibility of identifying the true culprit by narrowing down the range of suspects. Although many techniques based on mitochondrial DNA (mtDNA) have been developed, challenges remain to cost-effectively identify species from degraded samples containing a mixture of DNA from multiple species and to standardize procedures for mammalian species identification. This review evaluates the reliability and versatility of mtDNA-based techniques to reveal obstacles to the establishment of standard analytical methods, with a particular focus on DNA mixtures. When samples contain a mixture of DNA from multiple species, the interpretation of sequencing analysis results is difficult. Although DNA metabarcoding using next-generation sequencing (NGS) technologies can overcome the DNA mixture problem, DNA metabarcoding is not suitable for the type of small-scale analysis routinely performed by local forensic laboratories, primarily because it is costly and time-consuming. By contrast, fluorescent multiplex PCR analysis enables cost-effective and simultaneous species identification from suboptimal samples, although the number of identifiable species is currently limited in comparison with sequencing techniques. The advantages and limitations of current techniques presented in this review indicate that multiplex PCR analysis will continue to be important for mammalian species identification in forensic casework analysis. Further developments in multiplex PCR analysis that enable the identification of an increased number of species will play a key step for standardization efforts among forensic laboratories.
... Analyses of dog mtDNA in forensic casework mostly target the non-coding control region (CR) or D-loop [5][6][7][8][9][10] because of its high variability. The CR region is about 1200 bp long and comprises two hypervariable regions (HV-I from position 15458 to 16129 and HV-II from position 16430 to 16727 relative to the Kim et al. [11] reference sequence) that are separated by a Variable Number of Tandem Repeats (VNTR) with a 10 bp motif. ...
... DNA based wildlife forensics is becoming a powerful tool aiding in identifying specimens according to their biological species [1]. DNA based species identification has been used in several cases to investigate the illicit hunting of animals and trade in protected species [2,3]. These methods have several advantages over conventional approaches for species identifications, especially in situations where identification methods based on morphological characters are not possible because only highly processed, degraded or trace amounts of specimen materials are the only items are available for identification purposes [4]. ...
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An animal skin confiscated from a suspect was referred to our laboratory for DNA analysis. The investigating officer suspected that skin may have been from a tiger cub, Panthera tigris. As this tiger species is listed under the Indian wildlife protection act (1972), possession or trading of such a skin would be a serious offence. However, according to Indian law, the investigating officer has only a limited amount of time to document the species of origin of the skin, and the evidence must be presented in a way that is acceptable in a court of law. Using a DNA barcoding approach based on DNA sequences from the CO1 gene, we were able to show within the one day that the animal skin was actually the skin of a cow (Bos indicus) that had been altered to appear as a tiger skin. Although DNA barcoding is not a novel application of a method for species identification, the rapid turnaround time needed to obtain conclusive results in such cases is an important factor that brings novelty to this method in forensic investigations. Using this DNA based analysis, a case statement was prepared for the identified confiscated skin. Ultimately, the investigating authority requested withdrawal of the case from consideration under the wildlife protection act.
... Usually, the two hypervariable regions (HV-I: positions 15458-16129; HV-II: 16430-16727; relative to the Kim et al. [2] reference sequence) of the non-coding control region (CR) are analyzed to associate a shed hair with a particular dog [3][4][5][6][7][8]. Although the CR is highly variable in dogs, about half of the 214 dogs from a Belgian population study have CR haplotypes Be47, Be23 and Be19 [9,10], which belong to the CR haplogroups B1, A17 and A11 (named after [11]). ...
... While DNA technologies have been routinely used to identify humans and provide evidence for crime since 1985 (Gill et al. 1985;Jeffreys et al. 1985a;1985b;Reeder 1999), their application to provide evidence for wildlife crime is still in its infancy. DNA can be extracted from degraded or highly processed products that are commonly encountered in illegal wildlife trade, such as from cooked meat (Martinez and Danielsdottir 2000), powdered bone (Prado et al. 2002), claws left on tanned hides (Hedmark and Ellegren 2005), egg shells (Moore et al. 2003), hair (Jedrzejewksi et al. 2005;Savolainen and Lundeberg 1999) and feathers (Rudnick et al. 2007). Information can be obtained on what species or composite of species the seizure has been derived from (Chapman et al. 2003;Hsieh et al. 2003;Huang et al. 2003;Ludwig 2008). ...
... Within the last decade, the scientific community has relied increasingly on DNA profiling to connect victims and suspects to crime scenes. Some of the techniques available today include the polymerase chain reaction (PCR) to amplify miniscule amounts of DNA for subsequent analyses (19)(20)(21)(22)(23)(24)(25), restriction fragment length polymorphisms (RFLP) (1,26), DNA sequencing, random amplified polymorphic DNA (RAPD) (27) and short tandem repeat (STR) analysis (1,(28)(29)(30). These DNA techniques can be used to create species-specific fingerprint databases to improve the reliability of the identification process, distinguish among very closely related species (25)(26)(31)(32)(33)(34)(35)(36) and may help eliminate misidentification or prevent the loss of information from larvae that die during rearing (34). ...
Article
Full-text available
Larvae of Diptera are used routinely in forensic entomology to estimate accurately the post mortem interval. These applications require a detailed understanding of insect growth rates and insect succession profiles under different climactic conditions and geographical locations. There may be times when larvae may have been removed from a single food source or the corpse has been moved from one location to another, confounding the interpretation of the entomological data. We report here a study that evaluates the time period during which a food source can be detected in larvae of Protophormia terraenovae. We could detect the food source (pork or chicken liver) that larvae had ingested from early second instar to late third instar larvae. No detection was possible in pupae or adults. When larvae were transferred from one food source to another, the primary source was detectable for only 8–12 hours after transfer, whereas it was detectable for 12 hours in starved larvae. In most studies that use insects to estimate PMI, investigators collect larvae directly from the corpse. In cases where there have been significant disturbances, investigators must understand the changes in insect dieting and development that may take place.
... Breeds may have significant founder effects, thus are always subject to sub-structuring. DNA mutation rate estimates of mtDNA CR DNA are also different between species [52][53][54][55][56][57][58], leading to higher or lower exclusionary power for a given mitochondrial region. The dog dataset was anticipated to require a smaller sampling compared to humans since the domestic dog mtDNA CR contains fewer polymorphisms [59] and mitotypes and provides less power of exclusion than humans [60]. ...
... Given the condition of material collected for examinations, the only solution is often to analyze mtDNA, since it can be amplified even if there are only small amounts of DNA or it is degraded (9). Although a greater emphasis is placed on the analysis of mtDNA from human material, canine mtDNA has occasionally been analysed in criminal and civil proceedings (1,2,13,15). To date, the results of analyses of the control region of the mtDNA D-loop from domestic dogs have been reported from such countries as Austria (5), Japan (16), Sweden (14), Turkey (6), and the United States (8). ...
Article
The aim of the study was to determine differences at the molecular level between different breeds of dogs on the basis of a comparative analysis of the mtDNA D-loop sequence. The material used in the study consisted of blood collected from 40 Doberman Pinscher, Dachshund, German Shepherd, and Crossbreed dogs. The investigations involved DNA extraction, amplification and sequencing of the mtDNA D-loop, as well as bioinformatic analyses. Fifteen SNP (single nucleotide polymorphism) mutations were identified. This study proposes a new solution for dog breed identification, i.e. the use of SNPs specific to each breed. The analysis revealed the presence of already known SNPs in various configurations depending on the breed. Breed-specific SNP patterns were identified in 94% of the Doberman dogs, and two SNP patterns were found in 73% of the German Shepherd dogs. This subject requires further research. However, given the limited amount of data on the use of mtDNA in determination of breed affiliation, the present results seem to be promising.
... Forensic scientist can provide effective information to courts through this framework to avoid providing confusing and unclear conclusion. Although a systematic study on forensic recognition had been carried out in the 20 th century, especially in the fields of gunshot, fingerprint, glass, tool marks and footprint [1][2][3][4][5][6], how to provide scientific evidence for courts is still a hot debate in many forum of law and forensic science. One reason is that the U.S. Supreme Court promulgate Daubert rulings [7] about the admissibility of evidence in 1993. ...
... Species characterization based on genetic identification has been achieved to study unlawful tracking of interested animals species 14,36 , illegal trading of endangered species in natural reserves 5,34 and residual of animal tissues in criminal cases 27 . This universal decrease in genetic resources of animal species may lead to several obstacles and restrictions in perspective breeding program to enhance the genetic composition of animal resources in addition to loss of desired genetic traits. ...
Article
Full-text available
We analyzed the genetic variability of Egyptian chicken breeds through mitochondrial D-loop region patterns. The current results found that eleven nucleotide sequences (ACTACGGGAAC) were observed only in all individuals of both of Egyptian chicken breed at the beginning of d-loop region of mitochondrial DNA. The results showed that the D-loop region of mitochondrial DNA has reliable polymorphisms as single nucleotide polymorphisms (SNPs) in the two Egyptian traditional chicken breeds. The phylogenetic tree revealed that both Fayoumi and Dandarawi shared same cluster and they are close to domesticated chicken species such as Gallus gallus spadiceus and Gallus gallus bankiva respectively with tiny distance in case of the wild type of Gallus lafayetii. A total number of eleven haplotypes observed in both of Egyptian chicken breed and nine polymorphic site were found in Egyptian chicken. The estimated haplotype diversity was 1.00±0.063 (Fayoumi breed) and 0.857±0.10 (Dandarawi breed) whereas the nucleotide diversity was 0.00398±0.0017 for Fayoumi and 0.00313±0.0015 in case of Dandarawi.
... DNA based wildlife forensics is becoming a powerful tool aiding in identifying specimens according to their biological species (1). DNA based species identification has been used in several cases to investigate the illicit hunting of animals and trade in protected species (2,3). These methods have several advantages over conventional approaches for species identifications, especially in situations where identification methods based on morphological characters are not possible because only highly processed, degraded or trace amounts of specimen materials are the only items are available for identification purposes (4). ...
Article
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An animal skin confiscated from a suspect was referred to our laboratory for DNA analysis. The investigating officer suspected that skin may have been from a tiger cub, Panthera tigris. As this tiger species is listed under the Indian wildlife protection act (1972), possession or trading of such a skin would be a serious offence. However, according to Indian law, the investigating officer has only a limited amount of time to document the species of origin of the skin, and the evidence must be presented in a way that is acceptable in a court of law. Using a DNA barcoding approach based on DNA sequences from the CO1 gene, we were able to show within the one day that the animal skin was actually the skin of a cow (Bos indicus) that had been altered to appear as a tiger skin. Although DNA barcoding is not a novel application of a method for species identification, the rapid turnaround time needed to obtain conclusive results in such cases is an important factor that brings novelty to this method in forensic investigations. Using this DNA based analysis, a case statement was prepared for the identified confiscated skin. Ultimately, the investigating authority requested withdrawal of the case from consideration under the wildlife protection act.
... 样品的采集和 DNA 的提取是科学研究中重要的 技术之一, 特别是对于一些取样比较困难的研究对 象, 如稀有的动物或保护类动物, 常用的取样方法有 3 种, 即伤害性取样、 非伤害性取样和非损伤性取样 [1] 。 伤害性取样即通过杀死动物而获得新鲜的肌肉、 肝脏 和血液等组织样品; 非伤害性取样是通过捕获动物来 抽取血液、 采集毛发或羽毛、 耳、 尾、 趾等样品; 非损伤 性取样法即在不触及或伤害动物本身的情况下, 通过 收集脱落的毛发或羽毛、 粪便、 尿液、 食物残渣、 鹿角、 鱼鳞和卵壳等不同形式的样品进行遗传分析的一种 取样方法 [2] 。自 1975 年 Bradfied 等成功地从头发中提 取 DNA 以来, 毛发已成为法医学、 遗传学常用的检测 材料之一 [3] , 它具有无创伤性、 取材痛苦小、 运输和储 存方便、 可靠、 重复性高等优点 [4] 。粪便样品中含有大 量的食物残渣及各种消化道成分, 因此粪便中提取 DNA 要比血液和组织中困难得多,所以此法仅用于濒 危动物 [5] ...
Article
Full-text available
37 卷第 8 期 总第 509 期 样品的采集和 DNA 的提取是科学研究中重要的 技术之一, 特别是对于一些取样比较困难的研究对 象, 如稀有的动物或保护类动物, 常用的取样方法有 3 种, 即伤害性取样、 非伤害性取样和非损伤性取样 [1] 。 伤害性取样即通过杀死动物而获得新鲜的肌肉、 肝脏 和血液等组织样品; 非伤害性取样是通过捕获动物来 抽取血液、 采集毛发或羽毛、 耳、 尾、 趾等样品; 非损伤 性取样法即在不触及或伤害动物本身的情况下, 通过 收集脱落的毛发或羽毛、 粪便、 尿液、 食物残渣、 鹿角、 鱼鳞和卵壳等不同形式的样品进行遗传分析的一种 取样方法 [2] 。自 1975 年 Bradfied 等成功地从头发中提 取 DNA 以来, 毛发已成为法医学、 遗传学常用的检测 材料之一 [3] , 它具有无创伤性、 取材痛苦小、 运输和储 存方便、 可靠、 重复性高等优点 [4] 。粪便样品中含有大 量的食物残渣及各种消化道成分, 因此粪便中提取 DNA 要比血液和组织中困难得多,所以此法仅用于濒 危动物 [5] 。 目前, 鸡的 DNA 提取多采用血液样品, 而对于羽 毛样品基因组 DNA 提取少见详细报道。本研究通过 对血液、 肝脏和羽毛不同部位的样品进行不同程序的 处理, 并对提取基因组 DNA 进行了应用效果的分析, 获得了有效且伤害性小的方案, 可用于提取高质量的 基因组 DNA, 为禽类分子生物学方面的研究提供更广 阔的样本来源和技术方法。 1 材料与方法 1.1 试验动物 鸡来自辽宁省畜牧科学研究院与沈阳农业大学 养殖实验场。 摘 要: 采用伤害性取样和非伤害性取样, 采集鸡的肝脏、 血液、 羽髓、 羽鞘、 羽根、 实心羽茎和羽 毛上脐周围绒羽等不同部位样品, 并采取对样品不同处理程序提取基因组 DNA。结果表明, 鸡的微 量血液 (50 μl) 和羽髓样品提取基因组 DNA 与肝脏 (50 mg) 样品提取的基因组 DNA 的量相当; 羽鞘、 羽根、 实心羽茎和羽毛上脐周围绒羽可以提出一定量的基因组 DNA, 其提取量少于肝脏和羽髓组织, 但可以满足各种涉及 PCR 反应的分子生物学试验, 肝脏羽髓消化最适时间为 2.5 h, 其他样品需消化 过夜。该研究结果为禽类分子生物学研究拓宽了样本来源。 关键词: 鸡; DNA 提取; 伤害性取样; 非伤害性取样 Abstract: Liver, blood, feather pulp, theca of feather, quill, solid rhachis, and plumila of upper umbili• cusand different parts of feather samples of chicken were sampled by destructive and nondestructive sampling for the DNA isolation. The different treating protocols were used for the isolation. The results showed that the yields and qualities of DNA isolated from blood (50 μl) and feather pulp are nearly to livers', and those from theca of feather, quill, solid rhachis and plumila of upper umbilicus are some less but it is suitable to the experiment of molecular biology involved PCR. The protocol of DNA isola• tion provides new sources of samples for ornithic molecular biology study. Key words: chicken; DNA isolation; destructive sampling; nondestructive sampling 55
... 样品的采集和 DNA 的提取是科学研究中重要的 技术之一, 特别是对于一些取样比较困难的研究对 象, 如稀有的动物或保护类动物, 常用的取样方法有 3 种, 即伤害性取样、 非伤害性取样和非损伤性取样 [1] 。 伤害性取样即通过杀死动物而获得新鲜的肌肉、 肝脏 和血液等组织样品; 非伤害性取样是通过捕获动物来 抽取血液、 采集毛发或羽毛、 耳、 尾、 趾等样品; 非损伤 性取样法即在不触及或伤害动物本身的情况下, 通过 收集脱落的毛发或羽毛、 粪便、 尿液、 食物残渣、 鹿角、 鱼鳞和卵壳等不同形式的样品进行遗传分析的一种 取样方法 [2] 。自 1975 年 Bradfied 等成功地从头发中提 取 DNA 以来, 毛发已成为法医学、 遗传学常用的检测 材料之一 [3] , 它具有无创伤性、 取材痛苦小、 运输和储 存方便、 可靠、 重复性高等优点 [4] 。粪便样品中含有大 量的食物残渣及各种消化道成分, 因此粪便中提取 DNA 要比血液和组织中困难得多,所以此法仅用于濒 危动物 [5] ...
Article
Full-text available
采用伤害性取样和非伤害性取样,采集鸡的肝脏、血液、羽髓、羽鞘、羽根、实心羽茎和羽 毛上脐周围绒羽等不同部位样品,并采取对样品不同处理程序提取基因组 DNA。结果表明,鸡的微 量血液(50 μl)和羽髓样品提取基因组DNA与肝脏(50 mg)样品提取的基因组DNA的量相当;羽鞘、 羽根、实心羽茎和羽毛上脐周围绒羽可以提出一定量的基因组 DNA,其提取量少于肝脏和羽髓组织, 但可以满足各种涉及PCR反应的分子生物学试验,肝脏羽髓消化最适时间为2.5 h,其他样品需消化 过夜。该研究结果为禽类分子生物学研究拓宽了样本来源。
... In such cases, distinguishing between wolf and dog is valuable (Sundqvist et al. 2008; is it also important to prove cases of illegal killing or poaching (e.g. Savolainen & Lundeberg 1999), or to identify hybrids (Vilà et al. 2003b). Individual identification (Q2) using genetic profiling of samples, such as saliva left on a kill, permits the identification of one or multiple problematic individuals and their sex (Sundqvist et al. 2008. ...
Article
p>Following protection measures implemented since the 1970s, large carnivores are currently increasing in number and returning to areas from which they were absent for decades or even centuries. Monitoring programmes for these species rely extensively on non-invasive sampling and genotyping. However, attempts to connect results of such studies at larger spatial or temporal scales often suffer from the incompatibility of genetic markers implemented by researchers in different laboratories. This is particularly critical for long-distance dispersers, revealing the need for harmonized monitoring schemes that would enable the understanding of gene flow and dispersal dynamics. Based on a review of genetic studies on grey wolves Canis lupus from Europe, we provide an overview of the genetic markers currently in use, and identify opportunities and hurdles for studies based on continent-scale datasets. Our results highlight an urgent need for harmonization of methods to enable transnational research based on data that have already been collected, and to allow these data to be linked to material collected in the future. We suggest timely standardization of newly developed genotyping approaches, and propose that action is directed towards the establishment of shared single nucleotide polymorphism panels, next-generation sequencing of microsatellites, a common reference sample collection and an online database for data exchange. Enhanced cooperation among genetic researchers dealing with large carnivores in consortia would facilitate streamlining of methods, their faster and wider adoption, and production of results at the large spatial scales that ultimately matter for the conservation of these charismatic species.</p
... For instance, more sub-haplogroups within haplogroups D and F were identified in DomeTree (Table S3). The improved tree would also facilitate mtDNA forensic studies in grey wolf [18,19]. ...
... In such cases, distinguishing between wolf and dog is valuable (Sundqvist et al. 2008; is it also important to prove cases of illegal killing or poaching (e.g. Savolainen & Lundeberg 1999), or to identify hybrids (Vilà et al. 2003b). Individual identification (Q2) using genetic profiling of samples, such as saliva left on a kill, permits the identification of one or multiple problematic individuals and their sex (Sundqvist et al. 2008. ...
Article
Full-text available
Following protection measures implemented since the 1970s, large carnivores are currently increasing in number and returning to areas from which they were absent for decades or even centuries. Monitoring programmes for these species rely extensively on non-invasive sampling and genotyping. However, attempts to connect results of such studies at larger spatial or temporal scales often suffer from the incompatibility of genetic markers implemented by researchers in different laboratories. This is particularly critical for long-distance dispersers, revealing the need for harmonized monitoring schemes that would enable the understanding of gene flow and dispersal dynamics. * Based on a review of genetic studies on grey wolves Canis lupus from Europe, we provide an overview of the genetic markers currently in use, and identify opportunities and hurdles for studies based on continent-scale datasets. * Our results highlight an urgent need for harmonization of methods to enable transnational research based on data that have already been collected, and to allow these data to be linked to material collected in the future. We suggest timely standardization of newly developed genotyping approaches, and propose that action is directed towards the establishment of shared single nucleotide polymorphism panels, next-generation sequencing of microsatellites, a common reference sample collection and an online database for data exchange. * Enhanced cooperation among genetic researchers dealing with large carnivores in consortia would facilitate streamlining of methods, their faster and wider adoption, and production of results at the large spatial scales that ultimately matter for the conservation of these charismatic species.
... The study by Savolainen & Lundeberg looked at another case in which dog hairs were found in the vehicle of a suspect, that were believed to belong to the family dog of a missing female whose body or location had not been found. After typing the hairs in the vehicle compared to the female's dog, the hairs were found to come from two different dogs, and the suspect was excluded (Savolainen & Lundeberg, 1999). ...
... Genetic species identification have been used to investigate the illegal hunting of animals, trade in protected species and animal tissues in human murder cases [1]. When morphology is compromised, genetic species identification attempts to match an unknown evidence sample to a known reference sample by comparing sequences of genes. ...
Article
Species identification is an essential issue in forensic casework. The introduction of DNA barcoding highlighted the expanding use of Cytochrome c Oxidase I mitochondrial gene (COI) as a genetic marker for species identification. This study aims to look at the current method for species identification and how the COI methodology could be applied in a forensic environment. In particular, to test the effective application of COI analysis in forensics, we created experimentally critical conditions such as environmental and mixtures, commonly found in forensic cases, when stain morphology is often compromised.
Article
In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.
Article
We sequenced the entire ∼16 kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., F(ST)=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).
Article
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A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cattle, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard), even from a biological sample containing a DNA mixture of multiple species. The assay was designed to identify species through multiplex PCR and capillary electrophoresis, with a combination of amplification of a partial region of the mitochondrial D-loop by universal primer sets and a partial region of the cytochrome b (cyt b) gene by species-specific primer sets. The assay was highly sensitive, with a detection limit of 100 copies of mitochondrial DNA. The assay’s ability to identify species from complex DNA mixtures was demonstrated using an experimental sample consisting of 10 species. Efficacy, accuracy, and reliability of the assay were validated for use in forensic analysis with the guidelines of Scientific Working Group on DNA Analysis Methods (SWGDAM). The multiplex PCR assay developed in this study enables cost-effective, highly sensitive, and simultaneous species identification without massively parallel sequencing (MPS) platforms. Thus, the technique described is straightforward and suitable for routine forensic investigations.
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Approximately 81.7 million cats are in 37.5 million U.S. households. Shed fur can be criminal evidence because of transfer to victims, suspects, and/or their belongings. To improve cat hairs as forensic evidence, the mtDNA control region from single hairs, with and without root tags, was sequenced. A dataset of a 402-bp control region segment from 174 random-bred cats representing four U.S. geographic areas was generated to determine the informativeness of the mtDNA region. Thirty-two mtDNA mitotypes were observed ranging in frequencies from 0.6–27%. Four common types occurred in all populations. Low heteroplasmy, 1.7%, was determined. Unique mitotypes were found in 18 individuals, 10.3% of the population studied. The calculated discrimination power implied that 8.3 of 10 randomly selected individuals can be excluded by this region. The genetic characteristics of the region and the generated dataset support the use of this cat mtDNA region in forensic applications.
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Dogs worldwide share a single genetic origin from Asian wolves. While dog and wolf lineages are difficult to separate in terms of nuclear genes, mitochondrial lineages are clearly distinguishable for the two species, offering a good opportunity to evaluate the differences between them. Species identification from DNA is an important tool for wolf conservation in Croatia, and wolf - dog differentiation is necessary for forensic cases, wildlife management and scientific research. The goal of this paper was to provide a data set on Croatian dog and wolf mitochondrial DNA control region sequences, and to research if these sequences can be used as a reference for species identification. We analyzed 281 base pair sequences of the mitochondrial DNA (mtDNA) control region of 20 mixed breed dog blood samples, 91 grey wolf muscle samples and two muscle samples of wolf-like animals. We identified 12 dog and 4 wolf mtDNA control region haplotypes. None of the haplotypes were shared, confirming that mtDNA control region haplotypes can be used to discriminate between Croatian wolves and dogs, and to confirm the maternal ancestry of putative hybrids. The sequences of the two wolf-like animals clearly grouped into a dog cluster.
Article
The discriminatory power of the noncoding control region (CR) of domestic dog mitochondrial DNA alone is relatively low. The extent to which the discriminatory power could be increased by analyzing additional highly variable coding regions of the mitochondrial genome (mtGenome) was therefore investigated. Genetic variability across the mtGenome was evaluated by phylogenetic analysis, and the three most variable ~1 kb coding regions identified. We then sampled 100 Swedish dogs to represent breeds in accordance with their frequency in the Swedish population. A previously published dataset of 59 dog mtGenomes collected in the United States was also analyzed. Inclusion of the three coding regions increased the exclusion capacity considerably for the Swedish sample, from 0.920 for the CR alone to 0.964 for all four regions. The number of mtDNA types among all 159 dogs increased from 41 to 72, the four most frequent CR haplotypes being resolved into 22 different haplotypes.
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The use and convenience of canine tetranucleotide microsatellite markers for forensic purposes is addressed. These are commonly used in human genetics due to their high polymorphism, ease of laboratorial management and low mutation rates. However, care should be taken before including these markers in canine panels due to the high mutation rates shown in this species and the risk of errors in paternity assignment. To answer this question a panel including 16 markers (8 of them tetranucleotides) were typed for the family members of the Dogmap Project reference panel, and their mutation rates calculated. The effect these rates would have in terms of the increase in the number of false positives is as-sessed. Excluding one single marker with extreme mutation rate from the panel considerably improves the average rate and the performance of the panel. Therefore, a careful examination of the mutational behaviour of the candidate markers is advised for any marker panel design, even prior to the assessment of their polymorphism.
Article
The structural organization of the mitochondrial genome of dogs is briefly considered, paying more attention to its variable areas, including the control region and its hypervariable parts HV1 and HV2, as well as a block of tandem decameric repeats and a homopolymer tract of cytosines and thymines. It is noted that the detected polymorphism of mitogenomes forming the clades with haplotypes, with few exceptions, are practically unrelated to dog breeds and their geographical place of residence. In general, the polymorphism of the mitochondrial DNA of dogs does not allow unambiguously identifying a specific individual and in criminalistics, in most cases, it is possible with one or another probability, depending on the variable areas taken into analysis, only to exclude suspected dogs that could lead to their owners from further investigation. Moreover, this requires the creation of population databases of canine mitogenomes, which can be used to calculate the probability of coincidence of haplotypes. Examples of individual cases of investigation of crimes in which polymorphism of DNA of dogs were used, including those that ended with a conviction, are given. Attention is drawn to the need for wider introduction into forensic practice of usage of canine DNA, which can obtained from traces left by dogs mainly in the form of saliva or wool (individual hairs).
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Dogs worldwide share a single genetic origin from Asian wolves. While dog and wolf lineages are diffi cult Dogs worldwide share a single genetic origin from Asian wolves. While dog and wolf lineages are diffi cult to separate in terms of nuclear genes, mitochondrial lineages are clearly distinguishable for the two species, to separate in terms of nuclear genes, mitochondrial lineages are clearly distinguishable for the two species, offering a good opportunity to evaluate the differences between them. Species identifi cation from DNA is an offering a good opportunity to evaluate the differences between them. Species identifi cation from DNA is an important tool for wolf conservation in Croatia, and wolf -dog differentiation is necessary for forensic cases, important tool for wolf conservation in Croatia, and wolf -dog differentiation is necessary for forensic cases, wildlife management and scientifi c research. The goal of this paper was to provide a data set on Croatian dog wildlife management and scientifi c research. The goal of this paper was to provide a data set on Croatian dog and wolf mitochondrial DNA control region sequences, and to research if these sequences can be used as a and wolf mitochondrial DNA control region sequences, and to research if these sequences can be used as a reference for species identifi cation. We analyzed 281 base pair sequences of the mitochondrial DNA (mtDNA) reference for species identifi cation. We analyzed 281 base pair sequences of the mitochondrial DNA (mtDNA) control region of 20 mixed breed dog blood samples, 91 grey wolf muscle samples and two muscle samples of control region of 20 mixed breed dog blood samples, 91 grey wolf muscle samples and two muscle samples of wolf-like animals. We identifi ed 12 dog and 4 wolf mtDNA control region haplotypes. None of the haplotypes wolf-like animals. We identifi ed 12 dog and 4 wolf mtDNA control region haplotypes. None of the haplotypes were shared, confi rming that mtDNA control region haplotypes can be used to discriminate between Croatian were shared, confi rming that mtDNA control region haplotypes can be used to discriminate between Croatian wolves and dogs, and to confi rm the maternal ancestry of putative hybrids. The sequences of the two wolf-like wolves and dogs, and to confi rm the maternal ancestry of putative hybrids. The sequences of the two wolf-like animals clearly grouped into a dog cluster. animals clearly grouped into a dog cluster.
Chapter
The forensic examination of animal hair is a well established discipline and has been so for over a century. The basis of all animal hair examination is microscopy, which may enable the hair analyst to identify a hair as animal in origin, characterize the hair to a particular family or species and to conduct comparative examinations. Animal hairs bear microscopic features or characteristics that may assist in their identification. These features can be external, such as the roots and the scale pattern present along the shaft of the hair and internal such as the medulla. Over the last century the examination of animal hairs remained essentially unchanged until the arrival of relative “newcomers” to the examination of animal hairs forum; the transfer and persistence of animal hairs; and DNA profiling. Preliminary studies conducted specifically on the transfer and persistence of animal hairs has shown that the results are comparable to the studies conducted on textile fibers. The advent of DNA profiling of animal hairs provided a degree of individualization which has not been possible with comparative microscopy.
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Dogs play an important role in terms of forensic science due to become reclaimable. It is effective incident resolution that detection of dog’s biological material at the crime scene on aggressive / victim material or detection of aggressive / victim material’s on dog’s material. The aim of this study analysis of HV-I and HV-II regions in mtDNA from Canis lupus familiaris blood samples and it demonstrate that it will be an important evidence source in illumination of the incident. For this purpose, after the isolation of the DNA target regions were amplified in the first PCR step, Exo-Sap method and ChargeSwitch ®-Pro PCR Cleanup kit purification was performed. The second stage of PCR v3.1 Cycle Sequencing kit was used and ABI Big Dye XTerminator and Sefadex method purification kit was performed. Index analysis was performed in the ABI3130 capillary electrophoresis instrument. Analysis of the raw data SeqScape 3.7 (Applied Biosystems), and the ClustalX, BioEdit programs was used. The results obtained in 1998, Kim et al. were analyzed by comparison with published reference number. It was established high polimorfic positions 15627, 15639, 15800, 15814, 15945, 15955, 16003, 16025 (HV-I region), 16431, 16439, 16495, 16611, 16619, 16624, 16637, 16658, 16672 (HV-II region). The results is converged of previous studies, 15 543 (4.03%) 15,640 (3.23%) 15,756 (6.45%), 15 945 (8.87), 15,951 (4.03%) , 15 960 (3.23%) in HV-I region, 16 495 (16.94), 16 585 (7.26%), 16 611 (15.32%), 16 619 (17.74%), 16 624 (12.90% ), 16 628 (4,84%), 16 635 (8.06%), 16,637 (11.29) 16,652 (4.84%) 16,654 (4.03%) 16,658 (13.71) 16,676 (4.03%) in HV-II region. 16 haplotypes have been identified in HV-I region; the distinction power is 0.98, the genetic diversity is 0.96. 12 haplotypes have been identified in HV-II region, the distinction power is 0.89, genetic diversity is 0.79. As a result, up to the number of haplotypes in HV-I region is identified and genetic diversity is high in this area, HV-I region is according to the members of each other the higher rate of distinction power to HV-II region. These polymorphisms are seen for the first time. This study was performed for the first time in Turkey, but in the field of forensic science methods used in international publications have been optimized according to different working hours and the cost is reduced. Performed with this arrangement is considered to contribute to the identification study.
Article
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Mitochondrial DNA can be isotopically labeled to the virtual exclusion of nuclear DNA labeling in cell lines lacking the major soluble thymidine kinase (EC 2.7.1.21) but retaining a mitochondrial thymidine kinase. This characteristic provides a means for the selective assay of mitochondrial DNA during isolation and a determination of the cellular content of mitochondrial DNA. The simplest of the three isolation procedures compared in this study is shown to yield approximately two-thirds of the total mitochondrial DNA in the tissue culture cells examined. Mouse L cells containing predominantly a 10⁷ dalton closed circular mitochondrial DNA species have 1100 ± 250 molecules per cell. A second line of mouse L cells which has mitochondrial DNA of molecular weight 2 x 10⁷ contains approximately 900 molecules per cell. HeLa cells have at least four times the mitochondrial DNA mass per mitochondrial volume as L cells.
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Nine skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmy at a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family.
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Mitochondrial DNA control region sequences were analyzed from 162 wolves at 27 localities worldwide and from 140 domestic dogs representing 67 breeds. Sequences from both dogs and wolves showed considerable diversity and supported the hypothesis that wolves were the ancestors of dogs. Most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. The sequence divergence within this clade suggested that dogs originated more than 100,000 years before the present. Associations of dog haplotypes with other wolf lineages indicated episodes of admixture between wolves and dogs. Repeated genetic exchange between dog and wolf populations may have been an important source of variation for artificial selection.
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A method has been developed for the direct sequencing of hypervariable region 1 (HV1) of domestic dog (Canis familiaris) and wolf (Canis lupus) mitochondrial DNA (mtDNA) using single hairs as template. The method uses a robotic work-station and an automated sequencer to allow for robust routine analysis. A population data base was created in order to investigate the forensic and population-genetic informativeness of domestic dog HV1. Sequence variation, partitioning of dog breeds among sequence variants and phylogenetic relations between the variants were determined. Samples from 102 domestic dogs of 52 different breeds and two captive wolves were analyzed. Nineteen dog-sequence variants were found and the frequencies of the variants ranged from 1 to 21%. The calculated discrimination power of the region, i.e., the exclusion capacity, implied that nine out of ten disputed individuals can be excluded by this analysis. The sequence variants were found to cluster into four phylogenetic groups.
Article
The identification of animal hairs is generally recognised to be one of the more difficult types of examination with which forensic scientists have to deal. The main reason for this is the variation that can exist within a species and even between hairs from the same animal. A considerable amount of experience is therefore required before the identification of animal hairs can be carried out with confidence. However, cases involving animal hairs are infrequent and experience is not readily acquired through casework alone.
Article
Mitochondrial DNA (mtDNA) was extracted from teeth stored from 3 months to 20 years, including teeth from the semi-skeletonized remains of a murder victim which had been buried for 10 months. Tooth donors and/or their maternal relatives provided blood or buccal cells, from which mtDNA was also extracted. Enzymatic amplification and direct sequencing of roughly 650 nucleotides from two highly polymorphic regions of mtDNA yielded identical sequences for each comparison of tooth and fresh DNA. Our results suggest that teeth provide an excellent source for high molecular weight mtDNA that can be valuable for extending the time in which decomposed human remains can be genetically identified.
Article
Several chemical and enzymatic properties were examined in the DNA extracted from dry remains of soft tissues that vary in age from 4 to 13,000 years and represent four species, including two extinct animals (the marsupial wolf and giant ground sloth). The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, which primarily manifest themselves as modifications of pyrimidines and sugar residues as well as baseless sites and intermolecular cross-links. This renders molecular cloning difficult. However, the polymerase chain reaction can be used to amplify and study short mitochondrial DNA sequences that are of anthropological and evolutionary significance. This opens up the prospect of performing diachronical studies of molecular evolutionary genetics.
Article
The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.
Article
Human hair follicles may be useful for determining individual differences in the metabolism of polycyclic aromatic hydrocarbons in epithelial tissues. for quantitative measurements of carcinogen metabolism, determination of the amount of DNA in the hair follicles is necessary in order to correct for individual size variation. A simple method for DNa determination in hair follicles is described, based on the use of a hypotonic pronase solution to solubilise DNA which is then available for complex formation with mithramycin or 4,6-diamidino-2-phenylindole. 2HCI (DAPI). The mithramycin method permits the measurement of DNA in a small number of hair follicles, while the DAPI method is sensitive for as little as one single bulb.
Article
A family exhibiting heteroplasmy at position 16355 in hypervariable region I of the human mtDNA control region has been identified. This family consists of a mother, daughter, and son. DNA samples extracted from blood stains, buccal swabs, and hairs from these individuals were amplified by PCR and sequenced utilizing fluoresence-labeled dye terminator chemistry in an automated DNA sequencer. In both the daughter and mother, heteroplasmy was observed in DNA extracted from blood stains, buccal swabs, and hairs. In the blood stains, the proportion of cytosine was greater than thymine in both individuals. Buccal swab extracts showed a more balanced contribution from the two nucleotides. Telogenic hair root and hair shaft samples exhibited a wide range of nucleotide contributions at this position, from predominately cytosine in some samples to predominately thymine in others. The apparent stochastic segregation of mitotypes in hair samples is discussed from a forensic viewpoint, and the mechanism of mtDNA heteroplasmy is considered.
Article
In the context of a study of wild chimpanzees, Pan troglodytes verus, we found that genotypes based on single PCR amplifications of microsatellite loci from single shed hair have a high error rate. We quantified error rates using the comparable results of 791 single shed hair PCR amplifications of 11 microsatellite loci of 18 known individuals. The most frequent error was the amplification of only one of the two alleles present at a heterozygous locus. This phenomenon, called allelic dropout, produced false homozygotes in 31% of single-hair amplifications. There was no difference in the probability of preferential amplification between longer and shorter alleles. The probability of scoring false homozygotes can be reduced to below 0.05 by three separate amplifications from single hairs of the same individual or by pooling hair samples from the same individual. In this study an additional 5.6% of the amplifications gave wrong genotypes because of contamination, labelling and loading errors, and possibly amplification artefacts. In contrast, amplifications from plucked hair taken from four dead individuals gave consistent results (error rate < 0.01%, n = 120). Allelic dropout becomes a problem when the DNA concentration falls below 0.05 ng/10 microL in the template as it can with shed hair, and extracts from faeces and masticated plant matter.
Article
This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Student's T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.
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tem for identification purposes. should read
S-100 44 Stockholm, Sweden tem for identification purposes. should read: J Forensic Sci Soc 1992;32:5-14.