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    Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism

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    Date
    2002-05-01
    Author
    Campbell, Rowan Stewart
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    Abstract
    Campbell, 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.
    Subject
    Equipment and Supplies
    Genetics
    Genetics and Genomics
    Genetic Structures
    Genomics
    Instrumentation
    Investigative Techniques
    Life Sciences
    Medical Genetics
    Medicine and Health Sciences
    Molecular Genetics
    Other Genetics and Genomics
    Probability
    mtDNA sequence analysis
    mitochondrial DNA polymorphism
    rapid analysis
    automatable virtual array screening system
    alternative methods
    sequence specific oligonucleotide hybridization
    single nucleotide polymorphisms
    Luminex 100 flow cytometer
    ABI Prism 310 Genetic Analyzer
    DNA analysis
    SNaPshot primer extension assay
    mtDNA haplotype
    SNP array
    URI
    https://hdl.handle.net/20.500.12503/29085
    Collections
    • School of Biomedical Sciences
    • Theses and Dissertations

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