DOI
https://doi.org/10.25772/eb9b-zw90
Defense Date
2021
Document Type
Directed Research Project
First Advisor
Dr. Baneshwar Singh
Second Advisor
Dr. D'Arcy P. Mays
Third Advisor
Dr. Tal Simmons
Fourth Advisor
Dr. Claire Cartozzo
Abstract
In forensic contexts, samples containing heavily fragmented DNA are commonly encountered. Compromised biological samples are especially prevalent in instances where human remains have been submerged in an aqueous environment for extended periods of time. Nuclear DNA is particularly vulnerable to the prolonged exposure to heat, moisture, and bacterial degradation that are prevalent in aquatic settings. Paired with the difficulty of recovering DNA from skeletal remains, which are often the only remaining component after the soft tissues have been stripped away, mitochondrial DNA (mtDNA) analysis serves as an invaluable alternative. In this multifaceted study, mtDNA analysis was performed on waterlogged bone samples to determine the capabilities and limitations of a proposed mtDNA workflow. The results originating from two bone sample types, scapula and rib, were compared, in addition to a comparison between a freshwater lentic system, Henleys Lake, and a freshwater lotic system, the James River. To compare the effectiveness of two extraction methods, the samples were extracted once using an organic phenol-chloroform method and once using the magnetic-bead based ChargeSwitch® gDNA Plant Kit. The bone specimens used in this study were collected approximately every 500 ADD, up to 4500 ADD, for the purpose of determining at which ADD interval mtDNA sequence recovery was no longer possible. After using gel electrophoresis as a preliminary qualitative assessment of the total DNA extracts, quantitative PCR (qPCR) was then used as both a quantitative and qualitative assessment of the mtDNA. Using the qPCR data, the analyses of covariance (ANCOVA) demonstrated that the lentic location and the rib bone type exhibited more extensive mtDNA degradation than their counterparts, and that mtDNA quantitation values significantly decreased as ADD increased. Additionally, ChargeSwitch proved to be most effective at recovering high quantities of mtDNA. Using only the ChargeSwitch extracts, mitochondrial sequencing was performed, demonstrating that while location did not affect the ability of the workflow to recover a sequence, rib bones were more likely to yield a sequence than scapulae. It was also found that with each 500 ADD increase, the likelihood of recovering a mitochondrial sequence decreased by 40%. In its entirety, this study and its proposed workflow can assist forensic analysts with recovering mitochondrial profiles from degraded, waterlogged bone samples originating from multiple aquatic settings. Ultimately, a successful yield of a mitochondrial sequence will help with identification efforts for unidentified skeletal remains.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
3-21-2021