When unknown remains are discovered in mass graves, such as those found in the war-torn nations of Vietnam, Kosovo, and Somalia, forensic researchers must overcome enormous obstacles. Circumstantial evidence and dental records are frequently inconclusive. Typically, DNA extracted from skeletal remains is significantly degraded, polluted, and present in very minute amounts. The low quality of these samples limits the effectiveness of conventional short tandem repeat (STR) typing procedures. In contrast, the benefits of mitochondrial DNA (mtDNA) sequencing make it an excellent tool for studying these samples. In contrast to nuclear DNA, it is abundant and found in hundreds of copies per cell. It has a low mutation rate. It is inherited from the mother and does not engage in sexual recombination, establishing maternal ties across generations. Forensic laboratories have been analyzing mtDNA using capillary electrophoresis (CE)-based Sanger sequencing for many years. The hypervariable I and II (HVI and HVII) sections are typically the targets of mtDNA sequencing for human identification, even though the whole mtDNA genome is just about 16 kb. These areas are contained in the “D-loop,” a 1.1 kb region.
Next-generation sequencing (NGS) devices enable mtDNA sequencing to be performed at high throughput, employing more straightforward procedures and capable of delivering deeper coverage of specific regions of interest. A fully integrated NGS tool, the Illumina MiSeq System offers a quick process and simplified library preparation that makes it perfect for sequencing tiny genomes and amplicon samples, such as the D-loop region of mtDNA. This application note explains how the MiSeq System is used to do deep sequencing of mtDNA’s HVI and HVII regions to help identify human skeletal remains discovered in mass graves in Vietnam. Cellular organelles called mitochondria transform the energy from food into a form that cells can utilize. Hundreds to thousands of mitochondria per cell are found in the fluid around the nucleus (the cytoplasm). Although mitochondria also contain some DNA, most are in chromosomes in the nucleus. “Mitochondrial DNA,” or “mtDNA,” refers to this genetic material. A minor portion of the total DNA in cells, mitochondrial DNA in humans comprises roughly 16,500 DNA base pairs. There are 37 genes in mitochondrial DNA necessary for healthy mitochondrial activity. Thirteen of these genes give instructions on how to create oxidative phosphorylation-related enzymes. Adenosine triphosphate (ATP), the primary energy source for cells, is produced by oxidative phosphorylation using oxygen and simple carbohydrates. The remaining genes give instructions for creating DNA’s chemical cousins, ribosomal RNA (rRNA) and transfer RNA (tRNA), respectively. These RNA types aid in assembling functional proteins from amino acid building blocks. Human bone mitochondrial DNA (mtDNA) research and the analysis of human bones using mtDNA technology may assist in identifying human skeletal remains and clarifying historical events. For instance, a recent tibia from a skeleton buried in Argentina for roughly 13 years, a fibula from Polynesia dating before 1778, and mtDNA was taken from human and animal bones and amplified by PCR showed that considerable amounts of genetic information might survive for long periods.
Overview of Mitotyping Protocol
The protocols used by the majority of mtDNA service providers are relatively similar. All samples, including DNA-rich blood reference samples, are treated with extreme care throughout the sampling and sequencing process. The approach’s primary goal is to keep pieces from contaminating one another. The analysis primarily relies on a PCR amplification technique that manages the hypervariable regions HVR-I and HVR-II. The primary visual analysis, sample preparation, DNA extraction, PCR amplification, post-amplification, quantification, purification, automated DNA sequencing, and data analysis are some of the phases in the mtDNA analysis process. Due to DNA damage or variations in the sequence, such as length heteroplasmy or, more particularly, unusual site heteroplasmy, the case samples necessitate special care during the PCR and sequencing steps.
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For example, Seven bone samples altogether were taken from the skeletal remains: six mid-shaft samples, each measuring around 25 mm in length, including pieces of the right humerus, right ulna, right radius, left tibia, left fibula, and left radius, as well as a 25 mm bone wedge from the right hip’s ischium. 3 to 15 g of bone was extracted from each sample. The bone shavings were kept at -70°.
All seven bone specimens had their DNA extracted following the technique. More than 2 g of bone was extracted. The bone was scrubbed vigorously to remove the outer layer of foreign material, broken into small pieces measuring about 0.3 cc, and then processed using a Tekmar Tissumizer to create a fine powder. The bone powder was put into a 15 mL conical tube and decalcified for 8 to 12 hours by being washed three times in 8 mL of 0.5 M EDTA, pH 7.5. DNA was extracted using 3 mL of prewarmed extraction buffer (10 mM Tris-HC1, pH 8.0, 100 mM NaCI, 2% sodium dodecyl sulfate, and ten mM EDTA) containing 0,5 mg/mL Proteinase K; the extraction was carried out at 56° for 12 to 18 hours. The powder was washed three times with sterile deionized water to remove excess EDTA. The extraction suspension’s Proteinase K concentration was increased to 1.0 mg/mL, and an additional 5 hours of incubation at 56°C followed. Unabsorbed bone powder was centrifuged into pellets, and the supernatant was then transferred into a sterile 15 mL conical tube.
A trace amount of phenol was extracted with ether after the extract had been deproteinized with chloroform, phenol, and isoamyl alcohol (25:24:1). The ether was then eliminated after 15 minutes of incubation at 56. The DNA was further purified using a membrane-based size exclusion method and an Amicon Centricon | 30 spin column. The DNA samples were evaporated to dryness in a vacuum microcentrifuge as an alternative to ethanol precipitation. The pellets from tile drying were reconstituted in 100 txt of TE buffer (10 mM Tris-base, pH 7.6, 1 mM EDTA). Centrifugation was used to pellet the DNA, which was then dried, resuspended in t00 gL of TE buffer, and washed with 70% ethanol. Agar gel electrophoresis, ethidium bromide staining, and UV spectrophotometry were used to measure the amount of DNA isolated from the bone samples.
Measurement of human DNA
For comparison with the bone DNA extracts, sheared human placental DNA standards containing 200, 100, 50, 25, 12.5, and 6.25 ng of DNA were utilized. After three minutes of boiling to denature the DNA, the samples were put on ice. Using Hybond-N nitrocellulose membrane, a Schleicher and Schuell Manifold lI Slot Blotter was put together, a vacuum was applied, and the standards were loaded in duplicate to bracket the bone extracts. In a Stratalinker 2400, the DNA was cross-linked to the membrane using UV light. The random primer technique was used to mark the human-specific single locus nuclear DNA probe M31. Prehybridization and hybridization were carried out according to the prior instructions. The amount of DNA was calculated by comparing the unknowns to the benchmarks.
The Cetus Corporation AmpliType HLA-DQ-alpha Forensic DNA Amplification and Typing Kit’s instructions were followed to amplify the HLA-DQ-alpha region but with the following changes. The following PCR parameters were used during the amplification on a Perkin Elmer-Cetus Gene Amp 9600 Thermal Cycler: 94 C for ten s, followed by 94 C for 10 s, 60 C for 10 s, and 72 C for 10 s for 32 cycles, followed by a ten rain soak at 72 C. Cetus premix, 25 L of 25 mM magnesium chloride, and DNA sample were all added in the reaction mixes. Using agarose gel electrophoresis to evaluate the PCR result, the HLA-DQ-alpha type was identified following the method suggested by Cetus.
Polymerase Chain Reaction Amplification
The polymerase chain reaction (PCR) was used to amplify two portions of the mtDNA genome’s control region. The first segment covered nucleotides 121 to 340 (219 base pair (bp) product), and the second segment covered nucleotides 16140 to 16350 (210 bp product). The 219 bp product was amplified using the following conditions: 0.4 p, M F121 primer (5′-GCA GTA TCT GTC TTI” GAT TC, 121-140, 5′-end biotinylated by The Midland Certified Reagent Company), 0.2 mM dATP, dCTP, dTTP, and dGTP. F = forward), 0.4 p.M R340 (5′-GTG TTT AAG TGC
TGT GGC CA, 321-340, R – reverse), 0.2 mg/mL BSA, buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1, and 1.5 mM MgC12), and 6.25 units of Taq DNA polymerase (Cetus, Boehringer Mannheim Biochemical $’) in a 25 gL reaction volume. The amplification was performed for 32 cycles in a Perkin Elmer Cetus Gene Amp PCR 9600 ~ system: 94 C for 10 s, 50 C for 20 s, and 72 C for 45 s.
Recently, human remains have been identified using the tile mtDNA control region’s polymorphism properties. To directly explore polymorphisms in the hypervariable portions of the control region in DNA recovered from five-year-old skeletal remains discovered in a desert, utilized sequence-specific oligonucleotides (SSO). For each population studied (African, Asian, Caucasian, Japanese, and Mexican), the nine “hot sites” chosen for polymorphic variance yielded diversity values of at least 0.95, higher than HLA-DQ alpha and D1S80. Any SSO type over all nine regions would likely exclude 88.3 to 94.6% of the population, according to the likelihood of two unrelated people having the same SSO type being 1/19—1/8. The ability to distinguish between people based on mitochondrial SSO-typing is excellent, but when there is a shortage of DNA or the mtDNA to be studied is of poor quality, brief parts of the control area may yield information that is just as useful or even more so. The mtDNA sequence analysis, or SSO-typing of the mtDNA control region, is only effective as an identification tool if a pertinent sequence database is created and the validity of maternal inheritance is established. The sequence database that is currently accessible contains some data from isolated subpopulations. To report more acceptable frequency values, databases from the general population (Caucasians, Blacks, Hispanics, etc.) must be compiled. Additionally, there has been little research on the integrity of maternal inheritance of the DNA’s basic structure. Even though it is widely acknowledged that maternal inheritance is a reliable process, it would be beneficial to research to confirm maternal inheritance at the DNA sequence level for five or more generations within various maternal lineages.
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Anuwanshi Sharma is a researcher in the field of forensic science. She also contributes to various forensic websites as a guest writer.