Browsing by Subject "mtDNA"
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Item An assessment of qPCR assays for DNA concentration and degradation(2019-05) Cropper, Emily R.; Coble, Michael D.; Warren, Joseph E.; Phillips, Nicole R.Forensically challenged samples are often composed of degraded, damaged, or low template mitochondrial DNA (mtDNA). A real-time quantitative polymerase chain reaction (qPCR) assay can help determine if there is sufficient quantity and robust quality of mtDNA to move forward with downstream sequencing and analysis. The fundamental issue with qPCR is that the nominal quantity of the DNA calibrated along the commercial standard used for quantification can vary depending on the supplier and lot numbers. The National Institute of Standards and Technology (NIST) has developed a commercially available human DNA standard, Standard Reference Material (SRM) 2372a, which consists of nuclear DNA (nDNA) and mtDNA data on three wellcharacterized human genomic DNA preparations. The SRM 2372a was used to compare three qPCR assays: a non-commercial triplex assay, for mtDNA quantification, and two commercial assays, Quantifiler Trio (QFTrio) for nDNA quantification, and NovaQUANT for nDNA quantification and determination of the mtDNA/nDNA ratio. Quantification of the SRM uniformly across these three qPCR assays allowed for the conclusion that a robust, reproducible, accurate, and efficient qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the specificity of the qPCR chemistry, and (3) sound primers and probes, to name a few. The findings indicate that commercially available qPCR assays do not necessarily perform as marketed and should be re-verified by a validated DNA SRM.Item Comparison of Next Generation Sequencing Methodology on the Ion PGM™ System Performance versus that on the Sanger Sequencing Method for HV1 and HV2 Regions of mtDNA(2015-05-01) Argueta, Wendy C.; Arthur J. Eisenberg; Michael Allen; Raghu R. KrishnamoorthyAnalysis of mitochondrial DNA in forensic applications has enabled the identification of a missing person through comparison with additional maternal relatives. Most forensic applications are based on sequencing of both hypervariable regions of the mtDNA. Sequencing of these regions has been commonly done using Sanger-type sequencing (STS) methodology, which is expensive, time-consuming and laborious. Next Generation Sequencing (NGS) technology, such as the Ion Torrent PGM™ System platform, overcomes most of these issues. In this study, samples from the Guatemalan population (n=40) were sequenced with both Ion Torrent PGM™ technology and STS methods. A high level of consistency (98%) was observed among data derived from both methods. Most of the discrepancies were point heteroplasmy, which were more easily detected by PGM™ technology. In terms of performance, the NGS method was shown to be quick, with high-throughput and more efficient compared to the traditional STS method. More accurate and reliable sequencing data were obtained from the Ion Torrent PGM™ method due to its high level of coverage. Sequencing data for all individuals, representing 19 different family groups, were obtained using the NGS technology. Sequence polymorphisms were detected in 55 positions, from which 26 were observed only in relatives belonging to the same family and were not observed for any other family group. In a forensic context, haplotype specific polymorphisms are the basis for identification and comparison between evidence and reference samples purposes. Haplotypes between maternally related individuals were consistent in 18 family groups.Item Mitochondrial SOS: how mtDNA may act as a stress signal in Alzheimer's disease(BioMed Central Ltd., 2023-10-12) Gorham, Isabelle K.; Barber, Robert C.; Jones, Harlan P.; Phillips, Nicole R.BACKGROUND: Alterations in mitochondrial DNA (mtDNA) levels have been observed in Alzheimer's disease and are an area of research that shows promise as a useful biomarker. It is well known that not only are the mitochondria a key player in producing energy for the cell, but they also are known to interact in other important intracellular processes as well as extracellular signaling and communication. BODY: This mini review explores how cells use mtDNA as a stress signal, particularly in Alzheimer's disease. We investigate the measurement of these mtDNA alterations, the mechanisms of mtDNA release, and the immunological effects from the release of these stress signals. CONCLUSION: Literature indicates a correlation between the release of mtDNA in Alzheimer's disease and increased immune responses, showing promise as a potential biomarker. However, several questions remain unanswered and there is great potential for future studies in this area.Item Optimization of Filter Metrics for Mitochondrial DNA Sequence Analysis.(2009-08-01) Musslewhite, Pamela; Planz, John V.Quality metrics translate sequence information into numerical values which allows a software program to filter through data without human intervention. Primer specific settings for the trace score and contiguous read length in Sequence Scanner Software v1.0 (Applied Biosystems, Foster City, CA) were established using a calibration dataset of 2,817 sequence traces and validated using a second dataset of 5,617 sequence traces. Prior to optimization 51.7% of the samples required manual intervention while 28.4% require review after optimization. An evaluation of signal intensity and signal to noise ratio variables was performed and no trend was recognized for predictive modeling. Use of quality values per peak to ascertain confidence in the base call was evaluated and found to be a feasible parameter for sample quality assessment and confident base calling.Item The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit(2003-07-01) Cooksley, Joseph David; John Planz; Joseph Warren; Arthur EisenbergCooksley, Joseph David, The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit. Master of Science (Forensic Genetics), August 2003, 93pp., 3 tables, 14 figures, 1 appendix, references, 17 titles. Nucleotide sequences from coding regions in the mitochondrial DNA genome were evaluated for use in designing an oligonucleotide probe specific to humans that could be used with Quantiblot Human DNA Identification kit from Applied Biosystems. Two probes were designed and evaluated using the conditions, reagents, and protocols recommended in the kit instructions, as well as with less stringent conditions than those recommended in the kits instructions. Probe 1 bound to amplified mtDNA at low stringency conditions, with greater probe amount, with heat denaturation of the probe, and more HRP-SA enzyme conjugate added than recommended in the protocol. Probe 1 did not hybridize to mtDNA extracted from a buccal swab, bloodstain cards, or a whole blood sample. Probe 2 did not show any hybridization to mtDNA when evaluated.