DOI
https://doi.org/10.25772/81CS-0W37
Author ORCID Identifier
https://orcid.org/0000-0003-1526-0537
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Integrative Life Sciences
First Advisor
Tracey Dawson Green
Second Advisor
Sarah Seashols-Williams
Third Advisor
James Ferri
Fourth Advisor
James P Landers
Fifth Advisor
Evaldas Katilius
Abstract
The ever-increasing sexual assault evidence collection kit backlog within the United States has prompted the search (and desperate need) for a more efficient, cost-effective, and rapid processing technique that can separate male and female contributions from evidentiary samples. Although backlogs have largely been tackled across the United States and many technological advancements have made the forensic DNA analysis workflow quicker, techniques for handling sexual assault samples remain time-consuming, tedious, and inefficient at separating cellular fractions from the victim and perpetrator. Thus, this research sought to identify a reproducible sperm isolation method using antibodies and/or alternative differential cell lysis techniques as a possible solution. Automation of such techniques within a centrifugal microfluidic platform was also explored.
Although dithiothreitol (DTT) has been the primary reagent employed for the lysis of spermatozoa, results of studies herein revealed that residual DTT can artificially increase DNA quantities of various qPCR targets and impact predicted male-to-female DNA ratios. Thus, this research identified Promega’s Casework Direct kit, NP-40 cell lysis buffer, HGH (i.e., HTF media + glutathione + heparin), and alkaline (i.e., 1M NaOH) solution as promising direct-to-amplification lysis techniques which could generate sufficient DNA quantities and high quality STR profiles.
This research also developed a 35-minute sperm cell isolation assay using biotin-conjugated polyclonal PH-20 antibody and streptavidin-coated Microbubbles that could retain 58.0 ± 15% of seminal DNA within semen, as well as significantly and practically improve M:F ratios 2.76 ± 0.92-fold (p = 0.041) within STR profiles of processed mixture samples. Proof of concept studies demonstrated the feasibility of employing Microbubbles and buoyancy activated cell sorting (BACS) on a centrifugal microdevice platform, serving as the first assessment of BACS in such a format. Microbubbles were sufficiently mixed and aggregated, the position of Microbubbles within the microdevice was precisely controlled, and “subnatant” transfer was performed without significant loss of Microbubbles.
Finally, a modified differential cell lysis method using a combined prepGEMTM and alkaline approach was developed. With this assay, STR profiles of processed mixture samples exhibited 3.01 ± 2.3-fold improvement in M:F ratios and recovered 5.90 ± 7.8 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, the data indicated that using prepGEMTM enzyme and alkaline solution for lysis of non-sperm and sperm cells, respectively, could enrich for male DNA within sperm fractions and required only 25 minutes of overall processing time. Transition of this assay onto a centrifugal microdevice with a cell trap module served as proof of concept for accomplishing cell trapping and fractional separation in an automated format – which could be formatted for numerous biomedical and forensic applications.
Rights
© Brittany Celeste Hudson
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-15-2022