Specific quantification of human genomes from low copy number DNA samples in forensic and ancient DNA studies.
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We reviewed the current methodologies used for human DNA quantitation in forensic and ancient DNA studies, including sensitive hybridization methods based on the detection of nuclear alpha-satellite repetitive DNA regions or more recently developed fluorogenic real-time polymerase chain reaction (PCR) designs for the detection of both nuclear and mitochondrial DNA regions. Special emphasis has been put on the applicability of recently described different real-time PCR designs targeting different fragments of the HV1 mtDNA control region, and a segment of the X-Y homologous amelogenin gene. The importance of these quantitative assays is to ensure the consistency of low copy number DNA typing (STR profiling and mtDNA sequencing). (+info)
Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.
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AIM: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA). METHODS: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method. RESULTS: All 144 pairwise comparisons of HV1 and HV2 mtDNA fragments were successfully resolved by the DHPLC method. Forensic proficiency test standards were successfully resolved and DHPLC match/non-match results agreed with sequencing results provided by the test providers. The DHPLC method successfully identified one questioned sample that was prepared by the test provider as a body fluid mixture. MtDNA amplicon mixtures could be separated into their constitutive components by DHPLC and fraction collection approaches. CONCLUSIONS: DHPLC methods provide the forensic scientist with a powerful tool to rapidly screen mtDNA and may result in standardized methods to resolve mtDNA mixtures. These advances will allow mtDNA analysis in cases not previously examined by current sequencing-based approaches and could allow more forensic case samples to be entered into the proposed mtDNA Combined DNA Index System (CODIS trade mark ) databank as a result of mtDNA mixture resolution. (+info)
Identification of human remains by immobilized sequence-specific oligonucleotide probe analysis of mtDNA hypervariable regions I and II.
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AIM: A rapid analysis of mitochondrial DNA (mtDNA) sequences with an array of immobilized sequence-specific oligonucleotide (SSO) probes was tested on 18 skeletal elements recovered from mass graves in Croatia, which could not be genotyped with common forensic nuclear DNA systems (PM+DQA1 and short tandem repeat analysis). METHODS: We used duplex polymerase chain reaction (PCR) amplification of the mtDNA hypervariable regions I and II (HVI and HVII) (444 bp and 415 bp amplicons, respectively) and subsequent linear array typing, which targets six polymorphic regions and two additional sites within the human mtDNA HVI and HVII. The remaining amplified products were subjected to direct sequence analysis to obtain complete sequence information for the targeted HV regions. RESULT: Duplex PCR amplification of the mtDNA HVI and HVII was successful in providing sufficient product for typing with the array of SSO probes in 14 out of the 18 sample extracts. We report here the sequence match of one set of remains with a panel of immobilized SSO probes, followed by direct sequence analysis. The corresponding mtDNA haplotype obtained for the bone sample and the putative maternal reference was unique in a database of 105 randomly selected Croatian individuals. CONCLUSION: Mitochondrial DNA typing with an array of immobilized SSO probes can be a benefit to forensic DNA analysis of mass disaster remains and identity testing of single and mass graves. (+info)
Different dental tissues as source of DNA for human identification in forensic cases.
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AIM: To evaluate different dental tissues (pulp, dentin, and cement) as sources of DNA for forensic analysis. METHODS: A total of 20 teeth were obtained from unidentified bodies buried at the Central Cemetary in Bogota in 1995 and exhumed in 2000. The tissues from three teeth obtained after surgery were used as controls. The pulp cavity was exposed after cutting each tooth with a high-speed handpiece. The pulp was removed and processed separately from the dentin and cement. The dentin and cement were obtained by drilling with a high-speed handpiece and placed into separate tubes. DNA was extracted from mineralized tissues after a short decalcification step with EDTA. The DNA was quantified by dot-blot hybridization with D17Z1 probe, a primate- and human-specific alpha satellite DNA for the D17Z1 locus. Polymerase chain reaction was carried out for the hypervariable control region between nucleotides 29 and 408 bp (HV2 region) of the mitochondrial DNA, followed by gel electrophoresis to evaluate the amount and efficiency of the amplification. RESULTS: The pulp yielded the strongest amplification signals. The signals for dentin and cement were very similar because of the presence of cells of the periodontal ligament in the cement. The results of the amplification of the HV2 region of the mitochondrial DNA showed that dentin and cement acted as protective factors for the cells and allowed the conservation of the DNA. CONCLUSION: Cementoblasts and odontoblasts located within the cement and dentin are surrounded by the mineral matrix of the dental structure and thus protected from any environmental degradation forces, which makes them suitable sources for the DNA analysis. (+info)
How high should paternity index be for reliable identification of war victims by DNA typing?
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AIM: To analyze statistically and logically the significance of genetic matches between skeletal remains and relatives of missing persons in the process of identification of war victims by DNA typing. METHODS: DNA was isolated from bone and blood samples and short tandem repeat (STR) loci were typed by using AmpFLSTR Profiler, Profiler Plus, and Identifiler kits. Novel mini-haplotype analysis that compares matches in all three-locus combinations of alleles was developed and used in the analysis of inbreeding in the group of 295 unrelated individuals. RESULTS: While comparing 98 skeletal remains exhumed in the process of identification of war victims in Croatia with over 3,000 genotypes of relatives of missing persons, we revealed 20 cases of 14-locus matches and 4 cases of 15-locus matches between unrelated people. We hypothesized that this unexpectedly high number of false matches might be a consequence of local inbreeding and supported this hypothesis with very low correlation between the probability of a genotype and the number of matching genotypes in the database (R(2) = 0.36). Further support for the hypothesis was obtained by the analysis of mini-haplotypes, which revealed up to 90% overrepresentation of some mini-haplotypes. CONCLUSIONS: STR DNA typing is the "golden standard" of human identification, but evidential value of a genetic match can be easily misinterpreted. Therefore, careful use of statistical methods is essential for the proper evaluation of laboratory results. Whenever possible, multiple relatives should be analyzed and other evidence based on the information about time, place, and other conditions of disappearance, as well as anthropological and other "classical" forensic data should always be put together and compared before any final decision about the identity is made. (+info)
Applicability of 3D-CT facial reconstruction for forensic individual identification.
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Computed tomography (CT) is used in several clinical dentistry applications even by axial slices and two and three-dimensional reconstructed images (2D-CT and 3D-CT). The purpose of the current study is to assess the precision of linear measurements made in 3D-CT using craniometric patterns for individual identification in Forensic Dentistry. Five cadaver heads were submitted to a spiral computed tomography using axial slices, and 3D-CT reconstructions were obtained by volume rendering technique with computer graphics tools. Ten (10) craniometric measurements were determined in 3D-CT images by two examiners independently, twice each, and the standard error of intra- and inter-examiner measurements was assessed. The results demonstrated a low standard error of those measurements, from 0.85% to 3.09%. In conclusion, the linear measurements obtained in osseous and soft tissue structures were considered to be precise in 3D-CT with high imaging quality and resolution. (+info)
Forensic odontology: the roles and responsibilities of the dentist.
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Dentistry has much to offer law enforcement in the detection and solution of crime or in civil proceedings. Forensic dental fieldwork requires an interdisciplinary knowledge of dental science. Most often the role of the forensic odontologist is to establish a person's identity. Teeth, with their physiologic variations, pathoses and effects of therapy, record information that remains throughout life and beyond. The teeth may also be used as weapons and, under certain circumstances, may leave information about the identity of the biter. Forensic odontology has an important role in the recognition of abuse among persons of all ages. Dental professionals have a major role to play in keeping accurate dental records and providing all necessary information so that legal authorities may recognize malpractice, negligence, fraud or abuse, and identify unknown humans. (+info)
Human identification and analysis of DNA in bones.
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The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones. (+info)