Defense Date
2025
Document Type
Directed Research Project
First Advisor
Sarah Seashols-Williams
Second Advisor
Tracey Dawson Green
Third Advisor
Joseph Reiner
Abstract
Mixture interpretation is one of the more challenging aspects of forensic DNA analysis, due to many factors including unequal contributor ratios, allelic sharing, and allelic dropout. The traditional processing method for forensic mixtures containing sperm and vaginal epithelial cells is known as differential extraction, but this technique is time-consuming for analysts and requires extreme precision to avoid carryover from the opposite fraction. Differential extraction often still results in mixture profiles which then requires mixture deconvolution or the use of probabilistic genotyping. For this reason, alternate cell separation methods are being explored for a more precise separation at the front end of the workflow. This study evaluated the use of optical trapping, combined with a microfluidic device, for separation of sperm and vaginal epithelial cells from a postcoital mixture prior to traditional DNA processing. Previous work has demonstrated that optically trapping 40 sperm cells and 20 epithelial cells allows for generation of complete STR profiles with minimal carryover alleles, when processed traditionally. This research focused on the optical trapping of a postcoital sample to simulate a sexual assault evidentiary sample, which generated average profile completeness percentages of 69.8% for sperm fractions (average of 37 ± 6 sperm cells) and 43.2% for epithelial fractions (average of 11 ± 4 vaginal epithelial cells). However, four of nine sperm fractions (44%) and three of eight epithelial fractions (37.5%) generated profiles with more than 90% of expected alleles. One sperm fraction generated a complete STR profile with minimal female carryover. Additionally, direct amplification and single cell analysis were investigated as alternate DNA processing methods to determine how few cells can be processed for complete STR profiles. Although none of the samples directly amplified generated complete STR profiles, 10 trapped sperm cells generated ~50% completeness, and 10 trapped epithelial cells generated 87% completeness. The direct amplification results of 40 sperm cells and 20 vaginal epithelial cells fell short when compared to processing with traditional methods, as these same cell numbers yielded profile completeness percentages of 72.8 and 93.5%, respectively. Single sperm cells were also processed with direct amplification and generated a consensus profile of 78.3% from 20 sperm cells. Overall, optical trapping, combined with a microfluidic device shows strong potential in more efficiently separating cells from forensic mixtures for DNA processing, as compared to traditional methods. Additionally, implementation of an alternate processing method, such as direct amplification or single cell analysis, can decrease the time required for processing and reduce DNA loss, while allowing for STR profiles to be generated from fewer cells.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
12-9-2025