Defense Date
2023
Document Type
Directed Research Project
First Advisor
Dr. Christopher Ehrhardt
Second Advisor
Dr. Sarah Seashols-Williams
Third Advisor
Dr. William Eggleston
Abstract
The implementation of rapid DNA analysis systems is of interest to forensic DNA casework, as this technology exhibits a sample-to-profile time of approximately two hours with limited handling of samples. This decreases the likelihood of contamination, and can potentially reduce DNA backlogs; however, current rapid DNA analysis systems do not execute one crucial step of the forensic DNA analysis workflow: human DNA quantitation. This step is necessary for DNA profiles to be compliant with the FBI’s Quality Assurance Standard 9.4, allowing DNA profiles to be uploaded and searched within the CODIS database. Coupling traditional qPCR methods with rapid DNA analysis systems would be counterproductive due to the relatively slow turnaround times of qPCR. To address this issue, we tested flow cytometry as a method to rapidly estimate human DNA yield based on the quantity of cells in the sample. First, cell dilutions from various donors and bodily fluids (buccal tissue, whole blood, and whole saliva) were collected and subjected to flow cytometry to capture cell quantity and autofluorescence signatures. Next, four cell populations were derived from each cell dilution based on the captured autofluorescence signatures. Afterwards, the cell populations were subjected to DNA extraction and quantitation. Finally, cell quantity from all samples were compared to the respective human DNA yields to generate linear regression models assessing the relationship between cell quantity and human DNA yields across all dilutions and tissue types. Results showed that using cell quantity would provide uninformative estimates of DNA yield from blood, as regression models from blood dilutions displayed little to no relationships between cell quantity and DNA yield. The most promising predictions of DNA yield from cell quantities were exhibited within saliva, where three of the four individual donors exhibited strong positive correlations between cell quantities and DNA yields with one or more cell populations exhibiting coefficients of determination greater than 0.80. Buccal donors’ linear regression models also exhibited strong positive correlations between cell quantities and DNA yield across most donors and cell populations, with coefficients of determination ranging from 0.86-0.98. Two cell populations within buccal donors exhibited weak positive correlations between cell quantities and DNA yield: A24 “below” and “left” cells, with coefficients of determination of 0.17 and 0.59, respectively. When combining donors of the same tissue type in to a single linear regression model for each cell population, these measurements exhibited notable decreases across all tissue types. This method, with further research and validation, shows promise of quickly providing DNA quantitation values for evidence samples analyzed using rapid DNA analysis systems, allowing for the DNA profiles to be uploaded and searched within CODIS, along with assisting in decreasing the forensic DNA backlog.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-5-2023