Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing

In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region.In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries.In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.     

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.     

Forensic informativity of domestic dog mtDNA control region sequences

We have analysed the genetic information to be obtained from analysis of mitochondrial DNA (mtDNA) in domestic dogs studying the exclusion capacity in different populations and the correlation between mtDNA types and breeds or types of dogs. The exclusion capacities for a 573 bp sequence of the mitochondrial control region was between 0.86 and 0.95 for dogs in Sweden, the UK, Germany, Japan and China. The direct correlation between mtDNA type and breed, type of dog, and geographical origin of breed was generally low, but in some cases certain mtDNA types were overrepresented in one breed, and for wider groupings such as morphologically similar breeds, some mtDNA types were in many cases found in a distinct group of breeds, often originating from the same geographic region. This type of information may be used as an indication of the breed and, with some degree of probability, to include or exclude certain breeds from being the source of evidence materials.     

Mitochondrial Genome DNA Analysis of the Domestic Dog: Identifying Informative SNPs Outside of the Control Region*

Abstract: While the mitochondrial control region has proven successful for human forensic evaluations by indicating ethnic origin, domestic dogs (Canis lupus familiaris) of seemingly unrelated breeds often form large groups based on identical control region sequences. In an attempt to break up these large haplotype groups, we have analyzed the remaining c. 15,484 base pairs of the canine mitochondrial genome for 79 dogs and used phylogenetic and population genetic methods to search for additional variability in the form of single nucleotide polymorphisms (SNPs). We have identified 356 SNPs and 65 haplotypes in the remainder of the mitochondrial genome excluding the control region. The exclusion capacity was found to be 0.018. The mitochondrial control region was also evaluated for the same 79 dogs. The signals from the different fragments do not conflict, but instead support one another and provide a larger fragment of DNA that can be analyzed as forensic evidence.     

A population genetic database of cat breeds developed in coordination with a domestic cat STR multiplex

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.     

Application of hypervariable genetic markers to forensic identification of 'wild' from hatchery-raised red drum, Sciaenops ocellatus

Forensic identification of 'wild' versus hatchery-produced (cultured) red drum (Sciaenops ocellatus), an economically important marine fish in the southern United States, was assessed using hypervariable nuclear-encoded microsatellites and sequences of mitochondrial DNA. Both genotype exclusion and likelihood-ratio tests successfully identified 'wild' and 'cultured' individuals within requisite error bounds and within the context of complete parental sampling. Of the two, genotype exclusion was more effective, producing satisfactory results with fewer microsatellites and larger allowable error rates. Assignment tests proved ineffective, most likely because of the low level of genetic divergence between the sampled populations. An optimal, minimum set of ten markers that will reduce potential genotyping costs is identified. Results of the study should allay concerns regarding identification of 'wild'-caught fish sold illegally.     

Allowing for within-subpopulation inbreeding in forensic match probabilities

The importance of allowing for recent shared ancestry when calculating forensic match probabilities has been recognised for some time. Within-subpopulation inbreeding will also affect the match probability, and ignoring these effects can lead to an overstatement of the strength of the DNA evidence against a defendant. However, inbreeding can be readily accounted for in match probabilities by incorporating the inbreeding coefficient F_IS into calculations. We derive the necessary formulae, and demonstrate the effect of including F_IS (in addition to F_ST) in the equations.     

Forensic DNA against wildlife poaching: Identification of a serial wolf killing in Italy

The recent expansion of the Italian wolf population through the Apennine and western Alps, after centuries of contractions, is causing conflicts with human activities leading to a rise in poaching or illegal killings. Here we show how molecular population genetics has been used to identify a suspect serial wolf killer. We analysed DNA extracted from a necklace made of ten presumed wolf canine teeth, confiscated in 2008 to a man living in the northern Italian Apennine (Liguria Region). Individual genotypes were determined using 12 unlinked autosomal microsatellites (STRs), mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and three Y-linked STRs. Results indicate that the teeth belonged to six different individuals (three males and three females), which were assigned to the Italian wolf population with p > 0.90 by Bayesian procedures. One of these genotypes matched with the genetic profile of a male wolf previously found-dead and already non-invasively sampled in the same area. Another genotype matched with that of a female wolf non-invasively sampled twice in the same area 1 year before. These data are being used as forensic genetic evidence in the ongoing criminal trial against the suspect serial wolf killer.     

Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays

Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA that acts as an internal PCR control for detecting inhibition. Developmental validations were carried out following the revised guidelines of the Scientific Working Group on DNA Analysis Methods with modifications necessary for validation of nonhuman qPCR assays. All assays demonstrated the specificity, sensitivity, stability, reproducibility, accuracy, and precision required for forensic casework. The application of these assays to animal forensic DNA analysis has both conserved laboratory resources and improved genotyping results.     

DNA forensics and the poaching of wildlife in Italy: a case study

DNA molecular techniques were used in a forensic investigation involving the poaching of wildlife in a national park of Italy. A poacher, after having snared a wild boar (Sus scrofa) sow, knifed it to death. The animal was retrieved by conservation officers at the scene before the poacher could remove the carcass. Subsequently, the suspect denied the charges. During a search of his home, a bloodstained knife was confiscated. A method to identify the species from the DNA extracted from the stains revealed the blood to be that of the non-domestic form of Sus scrofa. Further DNA typing for individual identity using species-specific single tandem repeats or microsatellites (STRs) showed that the DNA on the knife matched that of the poached boar. Based upon the forensic evidence obtained, the suspect was convicted of poaching and of cruelty to animals.     

上、下尖牙髓腔/牙体表面积比值的年龄判定价值研究

目的 通过三维重建测量上、下颌尖牙髓腔/牙体表面积比值(Pulp/Tooth area Ratio,PTR)来推断个体年龄,为临床年龄推断的应用提供科学依据.方法 收集口腔门诊患者年龄在16～65岁200例锥形束CT(Cone beam computed tomography,CBCT)影像资料,以4个尖牙为测量对象并...     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Forensic DNA evidence and the death penalty in the Philippines

The death penalty remains a contentious issue even though it has been abolished in countries such as Australia, New Zealand, Canada, European Union member nations and some Asian countries such as Cambodia, East Timor and Nepal. Many argue that the irrevocability of the death penalty, in the face of potential erroneous convictions, can never justify its imposition. The Philippines, the first Asian country that abolished the death penalty in 1987, held the record for the most number of mandatory death offenses (30 offenses) and death eligible offenses (22 offenses) after it was re-imposed in 1994. Majority of death penalty convictions were decided based on testimonial evidence. While such cases undergo automatic review by the Supreme Court, the appellate process in the Philippines is not structured to accept post-conviction evidence, including DNA evidence.Because of the compelling nature of post-conviction DNA evidence in overturning death penalty convictions in the United States, different groups advocated its use in the Philippines. In one such case, People v Reynaldo de Villa, the defendant was charged with raping his 13-year-old niece that supposedly led to birth of a female child, a situation commonly known as ‘criminal paternity’. This paper reports the results of the first post-conviction DNA test using 16 Short Tandem Repeat (STR) DNA markers in a criminal paternity case (People v Reynaldo de Villa) and discusses the implications of these results in the Philippine criminal justice system.     

DNA evidence in jury trials: the "CSI effect"

In Murdoch v The Queen (2007) 167 A Crim R 329, Hillier v The Queen (2007) 228 CLR 618 and Forbes v The Queen (2009) 167ACTR 1, Australian appellate courts considered the interpretation of DNA evidence and the possibility of secondary transfer of DNA samples and questions about the statistical calculations used to produce probabilities of DNA matches. Following the 2010 Victorian case of Farrah Jama, whose conviction for rape was quashed 16 months into his prison sentence after it was discovered that the incriminating DNA sample was contaminated, Mr FRH Vincent QC, in his report to the Victorian Attorney-General, was scathing of the conduct of the case and made a number of recommendations, all of which were immediately adopted by the Victorian Government. Following the release of the Vincent Report, Australia's Attorneys-General have established a working party to examine national standards for the use and collection of DNA evidence. The use and interpretation of DNA evidence in jury trials is considered and factors that improve jury understanding of DNA evidence are discussed.     

Cell line DNA typing in forensic genetics--the necessity of reliable standards

The incorporation of reference DNA is crucial to the validation of any DNA typing protocol. This paper aims to provide a panel of reference DNAs for actual forensic profiling strategies, i.e. autosomal and gonosomal STR typing as well as mtDNA sequencing. We have characterised three human lymphoid cell lines, GM9947, GM9948 and GM3657, and considered 58 autosomal and gonosomal microsatellites as well as the mitochondrial control region sequence. Well-established markers and STRs recently developed for forensic use were involved. K562 DNA samples which we purchased from two different suppliers were also analysed. They revealed conflicting results with regard to the ChrX STR marker genotype. Hence, we suggest that K562 is no longer used for the calibration of profiling techniques. Our investigation establishes a panel of one female and two male DNA samples as an STR allelic ladder calibration tool and offers information on six alleles of each autosome (AS) marker, three alleles of each X chromosome (ChrX) marker and two alleles of each ChrY marker. In addition, sequences of the mitochondrial control region of the three DNAs are communicated in order to provide sequencing quality control.     

Extraction of DNA from decomposed human tissue. An evaluation of five extraction methods for short tandem repeat typing

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed biological material, a fact that has to be taken into account when choosing the appropriate casework methods. In this paper we report the evaluation of five different DNA extraction methods, namely the phenol-chloroform, the silica based, the InstaGene Matrix (BioTest), the glass fiber filter, and the Chelex based methods. The substrates for the analyses are decomposed human liver tissue specimens from forensic autopsy cases. Extracted DNA was quantified and DNA profiled by a set of seven STRs. We have compared laboratory time consumption and costs of the five methods, showing that the Chelex method is the more rapid and less expensive of the methods, the phenol-chlorophorm and silica extractions being the most time consuming and resource demanding ones. A full profile was obtained by the silica method in nine out of ten cases and this method failed to give a reliable type in four out of 70 STR analyses. The phenol-chlorophorm and the glass fiber filter methods failed in 16 analyses, the InstaGene Matrix (BioTest) in 25 and the Chelex extracts in 56 of the 70 STR analyses. By multiple logistic regression we show that the difference between the silica procedure and the other methods are statistically significant. In our hands, the silica gel extraction procedure is an obvious choice when the biological material available is decomposed human tissue--even if this procedure is one of the more laborious ones.     

Rapid genomic DNA extraction (RGDE)

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed as a biological material, a fact that has to be taken into account when choosing the appropriate casework methods. Nowadays expanded windows have been opened to the world especially in the area of genetic and biology science by performance of big projects such as human genome project. In this regard, one of the primary and important steps for all is DNA extraction with high quality and quantity in minimum time from biological material. By using RGDE method, genomic DNA with high quality and quantity can be acquired in the shortest time which has been presented in the world up to now. In this paper we report the evaluation of DNA extraction in this method.     

Canine Population Data Generated from a Multiplex STR Kit for Use in Forensic Casework*

Abstract: Canine biological specimens are often part of the physical evidence from crime scenes. Until now, there have been no validated canine‐specific forensic reagent kits available. A multiplex genotyping system, comprising 18 short tandem repeats (STRs) and a sex‐linked zinc finger locus for gender determination, was developed for generating population genetic data assessing the weight of canine forensic DNA profiles. Allele frequencies were estimated for 236 pedigreed and 431 mixed breed dogs residing in the U.S. Average random match probability is 1 in 2 × 10³³ using the regional database and 1 in 4 × 10³⁹ using the breed dataset. Each pedigreed population was genetically distinct and could be differentiated from the mixed breed dog population but genetic variation was not significantly correlated with geographic transition. Results herein support the use of the allele frequency data with the canine STR multiplex for conveying the significance of identity testing for forensic casework, parentage testing, and breed assignments.     

An STR forensic typing system for genetic individualization of domestic cat (Felis catus) samples

A forensic genotyping panel of 11 tetranucleotide STR loci from the domestic cat was characterized and evaluated for genetic individualization of cat tissues. We first examined 49 candidate STR loci and their frequency assessment in domestic cat populations. The STR loci (3-4 base pair repeat motifs), mapped in the cat genome relative to 579 coding loci and 255 STR loci, are well distributed across the 18 feline autosomes. All loci exhibit Mendelian inheritance in a multi-generation pedigree. Eleven loci that were unlinked and were highly heterozygous in cat breeds were selected for a forensic panel. Heterozygosity values obtained for the independent loci, ranged from 0.60-0.82, while the average cat breed heterozygosity obtained for the 11 locus panel was 0.71 (range of 0.57-0.83). A small sample set of outbred domestic cats displayed a heterozygosity of 0.86 for the 11 locus panel. The power of discrimination of the panel is moderate to high in the cat breeds examined, with an average P(m) of 3.7E-06. The panel shows good potential for genetic individualization within outbred domestic cats with a P(m) of 5.31E-08. A multiplex protocol, designed for the co-amplification of the 11 loci and a gender-identifying locus, is species specific and robust, generating a product profile with as little as 0.125 nanograms of genomic DNA.