Browsing by Subject "Instrumentation"
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Item Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism(2002-05-01) Campbell, Rowan Stewart; Eisenberg, Arthur J.; Budowle, Bruce; Planz, JohnCampbell, Rowan Stewart, Automatable Virtual Array Screening System For Rapid Analysis of Mitochondrial DNA Polymorphism. Doctor of Philosophy (Biomedical Sciences), May, 2002, 156 pp., 11 tables, 48 illustrations, bibliography, 96 titles. The goal of this research project was to develop alternative methods to traditional forensic mtDNA sequence analysis. Conventional forensic mtDNA analysis requires the direct sequencing of Hypervariable Region I and Hypervariable Region II in both the forward and reverse directions. This method is time consuming, labor intensive and expensive. Two methods for determining mtDNA haplotypes through the direct interrogation of Single Nucleotide Polymorphisms with HVI and HVII have been developed. A Sequence Specific Oligonucleotide Hybridization assay was developed on the Luminex 100™ flow cytometer, as well as a Single Base Extension assay developed for the ABI Prism® 310 Genetic Analyzer. The SNP typing of mtDNA sequences can provide a significant benefit in many forensic and human identification cases. The reassociation of mass disaster remains, mass grave analysis, and the screening of large numbers of crime scene samples are examples of their potential application. Their inclusion as a standard screening tool would be high beneficial since more extensive DNA analysis would be reserved for those samples that possess the greatest evidentiary value. In a blind study of 50 samples, the Sequence Specific Oligonucleotide Hybridization assay incorrectly identified the mtDNA haplotypes in 7 samples, whereas the Single Base Extension assay correctly identified each of the SNP positions interrogated. The SNaPshot™ primer extension assay was approximately 20-25 times more sensitive than the standard sequencing approach. This would suggest that this system could be a viable alternative to sequence analysis when samples are limited, as well as being more robust in detection and typing of heteroplasmic sites. A statistical evaluation of the SNP panels revealed that the genetic diversity estimated for the 50 Southwestern Hispanic samples tested was 0.9624 for the primer extension array and 0.9559 for the hybridization-based array. The probability of two randomly selected individuals from a population group having the same mtDNA haplotype was 0.0568 for the Single Base Extension assay and 0.0632 for the Sequence Specific Oligonucleotide Hybridization assay. A forensic mtDNA SNP array consisting of the positions evaluated in this study could provide a reasonable alternative to the full sequencing of the HVI and HVII regions.Item Validation of Applied Biosystems 3130xl Genetic Analyzer for Human mtDNA: An Evaluation of Three Amplification Series(2006-08-01) David, Jamalia Junelle; Joseph Warren; John Planz; Arthur EisenbergThe introduction of a new instrument into an accredited laboratory requires a documented internal validation. Validations typically include sensitivity, precision, and mixture studies. These tests assess the reliability and efficiency of the instrument and allow for interpretation guidelines to be established. This project consisted of a validation of Applied Biosystems’ 3130xl Genetic Analyzer and an evaluation of three mitochondrial DNA amplification primer sets for the control region. The validation was designed to evaluate the efficacy, robustness, and working limitations of the 3130xl instrument by performing a sensitivity study. A sensitivity study was performed using DNA sample dilutions, which were quantified using ABIs Quantifiler system to measure the amount of total nuclear DNA content in the samples. The samples were amplified on the GeneAmp PCR System 9700 in triplicate to evaluate stochastic activity. Three different primer sets were utilized which allow for the amplification of different regions of the human mitochondrial genome control region. After amplification, the quality and quantity of the DNA in all the samples was assessed using the Agilent 2100 BioAnalyzer, and subsequent sequence analysis was performed on the 3130xl Genetic Analyzer. Preliminary work has begun on a mixture study, but due to lack of time and reagents, this study was not completed and will have to be performed at a later date. All sequence data from the sensitivity study was evaluated using Sequencher version 4.1.4Fb19