The allele band patterns indicate that 5 of the skeletons belonged to a family group, comprising two parents and three children. The nine skeletons were further subjected to DNA typing using 5 STR markers: VWA/31, Thol, F13A1, FES/EPS, and ACTBP2. The sex of the remains were determined by amplifying the amelo-genin loci, and the results confirm the conclusion drawn from physical examination of the bones that the remains include four males and five female bodies. In 1992, scientists at the UK and the United States were requested to collaborate in the verification of the remains using DNA based techniques (Gill et al. In 1991, nine sets of skeletal remains were excavated from a shallow grave in Ekaterinburg, Russia, which were tentatively identified to include those of Nicholas II, Tsarina Alexandra, their three daughters, three of their servants, and the family doctor, Eugeny Botkin. The use of mtDNA sequence information to identify the 70-year old remains of the Russia Tsar Nicholas II and his family illustrates the power of DNA typing. ND4 / / Human mtDNA ND4L l l genome ND3 \\ 16,569 bp com * Labeled genes: ATP synthase (ATPase), cytochrome c oxidase (CO), cytochrome b (cyt b), NADH dehydrogenase (ND). The non-coding region includes the hypervariable regions, HV1 and HV2, used in DNA typing. The non-coding region (also known as the control region) has been estimated to vary about 1-3% between unrelated individuals, with the variations distributed throughout the HV1 and HV2 regions.įig. The sequence can then be compared to the available forensic database of human mitochondrial DNA sequences. The two hypervariable regions (HV1 and HV2) of the D-loop is amplified by PCR, providing sequence information for positions 16,024-16,30, respectively (Fig. The forensic value of mtDNA lies in the displacement loop (D-loop) of about 1,100 bp in length located in the non-coding region. Consequently, mtDNA information can reveal ancient population history and human evolution in anthropological investigations. Most importantly mtDNA comes solely from the mother through the mitochondria in her egg, and therefore, represents only the maternal ancestry of an individual. MtDNA has been extracted from teeth, hair shafts, bone fragments, all of which fail to yield forensic results with nuclear DNA markers. The likelihood of recovering mtDNA from very minute and degraded biological samples is greater than for nuclear DNA. The human mtDNA is circular with 16,569 bp (as opposed to the linear ~3 billion bp in the nuclear DNA), and it exists in hundreds to thousands of copies in a single cell. Mitochondria have an extranuclear DNA genome, the sequence of which was first reported for humans in 1981. MitoSAVE: mitochondrial sequence analysis of variants in Excel.In situations where nuclear (chromosomal) DNA typing is not an option (for example, insufficient quantities or too degraded), or an attempted typing using nuclear DNA markers is unsuccessful, mitochron-drial DNA (mtDNA) typing can be used. Table 2: DNA analysis software packages for NGS and/or Sanger data. Alternately, an Excel (Microsoft Corporation, Redmond WA) tool was developed by (King et al.) for converting variant calls from typical DNA analysis software to forensic mtDNA compliant nomenclature. Manufacturers of mtDNA analysis kits for the forensic market have developed solutions which incorporate appropriate alignment and nomenclature systems (see Table 2). Note that forensic nomenclature is not supported in most commercial software intended for routine DNA sequence analysis. BWA, SamTools, GATK) or through commercially available software, a non-exhaustive list of which is found in Table 2. The alignment of DNA sequencing data to the reference sequence (rCRS) can be achieved through many approaches, including open-source software (e.g.
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