Author ORCID Identifier
https://orcid.org/0009-0003-6550-5165
Defense Date
2024
Document Type
Directed Research Project
First Advisor
Sarah Seashols-Williams
Second Advisor
Tal Simmons
Third Advisor
Jenise Swall
Fourth Advisor
Ciara Rhodes
Abstract
Current methods for processing aged forensic bone samples for DNA analysis involve pulverization and organic extraction, which further destroys already degraded DNA and uses large amounts of limited samples. Additionally, the success rates for this type of analysis vary widely as homogenization incorporates PCR inhibitors into the sample. Little research has explored how pulverization results in DNA damage in profiles obtained from skeletal remains. This study proposes an enzymatic digestion method to separate osteocytes from PCR inhibitors in the extracellular bone matrix to improve the quality and quantity of DNA isolated from aged bone samples. The technique described here used demineralization followed by enzymatic digestion with collagenase I of bovine cortical bone slices as a model for aged human remains. These samples were then extracted with silica-based chemistry and quantified. The collagenase-digested samples had significantly lower yields of both bovine mtDNA and microbial 16S rDNA (p<0.001 for both). However, demineralized slices with no digestion yielded amounts of bovine mtDNA comparable to those from pulverized samples. Digestion with collagenase significantly decreased profile success (p<0.01) and quality compared to pulverized samples or demineralized slices. In contrast, demineralized slices had only slightly lower yields of bovine mtDNA but ultimately yielded equally successful profiles with greater profile integrity than pulverized bone samples. Eventually, this study concludes that collagenase I treatment did not improve the quality and quantity of DNA isolated from aged forensic bone samples and instead had a negative impact. However, demineralization alone offers a promising solution to the challenges of forensic bone analysis caused by the pulverization steps in the current workflow.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
4-28-2024
Included in
Forensic Science and Technology Commons, Musculoskeletal System Commons, Other Microbiology Commons