Identification of remains by sequencing of mitochondrial DNA control region

The maternity of two newborns who were murdered and abandoned >5 and 10 years were analyzed by amplification and direct sequencing of mitochondrial DNA (mtDNA) control regions. Sequences of two hypervariable segments from each femur bone sample and the blood of the putative mother showed four mutations in hypervariable region I and two mutations in addition to two nucleotide insertions in hypervariable region II compared with the reference sequence, and all sequences were identical. The genotype of these individuals is found to be relatively rare in the Japanese population, and it was strongly suggested that both sets of newborn remains really were children of the putative mother. Sexes of the remains were determined to be female and male by amplifying a segment of the X-Y homologous gene, amelogenin. These results demonstrate that sequencing of mtDNA is a useful tool for genetic identification of aged and decomposed materials.     

Extraction and amplification of authentic DNA from ancient human remains

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.     

Evaluation and identification of dismembered human remains.

On occasion, pathologists are confronted with cases involving dismembered human remains. Such cases present unique and interesting problems of postmortem identification. Four cases involving dismembered human remains are presented here and practical suggestions on how identification can be achieved are provided.     

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

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

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

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

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.     

Polymorphism of structural genes of human mitochondrial DNA for forensic medical personal identification

An experimental study is described, which deals with polymorphism of nucleotide sequences of three structural genes of mitochondrial genome, i.e. of the 3d, small 4th, and 6th subunits of the NADN dehydrogenase complex (ND3, ND4L and ND6) sampled from Russian population. The genetic primary structure was analyzed in 63 unrelated individuals. The investigated locuses were shown to possess a pronounced polymorphism. A total of 19 polymorphic positions were detected in the ND3, ND4L and ND6 gene region within the studied sampling. Besides, a possibility is demonstrated in the paper that the mtDNA structural genes can be used as additional identification markers in the forensic experimental typing of the mtDNA control region.     

Identification of decomposed human remains by deoxyribonucleic acid (DNA) profiling

After routine methods failed to establish positive identification of a decomposed homicide victim, deoxyribonucleic acid (DNA) typing techniques using blood from the victim and putative parents of the victim were used. This is the first report in the literature of a case using DNA fingerprinting in a "parentage" context to establish identity of unidentified, decomposed human remains.     

Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation

We describe a method combining in a single-round polymerase chain reaction amplifications of both cytochrome b and hypervariable D-loop mitochondrial DNA allowing species determination and individual human identification. Following the amplification step, amplicons are first screened on an agarose gel. The presence of only one band indicates that the sample is nonhuman, while the presence of two bands indicates a human origin. Subsequent DNA sequencing of the hypervariable D-loop region DNA allows for individual human identification as the presence of cytochrome b fragment does not interfere with the analysis. Similarly, further species determination on the basis of the phylogenetically variable cytochrome b gene is possible by sequencing of the cytochrome b DNA fragment.     

Recovery of human DNA profiles from poached deer remains: A feasibility study

Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA.We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles.Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to ‘drop in’ alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37 × 10^{− 3} to 9.53 × 10^{− 11}.This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to touch DNA in regards to simulated wildlife crime.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

DNA extraction and sex determination of human skeletal remains

DNA was extracted from human bones remained for 3 to 15 years by using the method developed in our laboratory. A pair of X-Y homologous primer were used to amplify the specific DNA fragments of Amelogenin gene. All the 35 samples yielded correct results. Our method is rapid, sensitive and reliable for the sex determination of skeletal remains.     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.     

线粒体DNA短片段复合扩增系统鉴别种属的研究

目的建立线粒体DNA短片段复合扩增体系用于种属鉴定的方法。方法提取人、牛、猪、羊、鸡的DNA,用所选的3对引物复合扩增细胞色素b基因（cyt b）片段、16srRNA基因片段和ND4基因片段,扩增产物经琼脂糖凝胶电泳检测。结果人DNA扩增产物在358bp、157bp和110bp处各出现一条带;动物DNA扩增产物均只有358bp一条带。结论线粒体DNA短片段复合扩增鉴别种属的方法可区分人源性生物检材和其它动物样本,可应用于法庭科学实践。