Author ORCID Identifier
https://orcid.org/0009-0008-2089-8539
Defense Date
2023
Document Type
Directed Research Project
First Advisor
Dr. Tracey Dawson Green
Second Advisor
Dr. Seyi Vanderpuye
Third Advisor
Sarah Schellhammer
Fourth Advisor
Dr. Brittany C. Hudson
Abstract
While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Previous work in the Dawson Green laboratory at VCU has developed alternative solutions for differential extractions with sexual assault samples using both an in-tube and microdevice assay. Prior work led to the identification of an alternative sperm lysis method – alkaline lysis using 1M NaOH. However, the current lysis method used for non-sperm cells, prepGEM™, is expensive and contains proprietary reagents, which may lead to future licensing complexities if used within the patented microdevice. Thus, the primary objective of this work was to find an alternative method that is inexpensive and non-proprietary for the lysis of non-sperm cells in sexual assault samples. Ideally, this would provide a quick and viable differential extraction technique for either in-tube or microdevice use.
In this study, non-sperm samples from five different donors were lysed with six reagents (alkaline buffer with 25-200mM NaOH, high salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer) in addition to the control method, prepGEM™. Quantification using Quantifiler® Trio for both vaginal and semen samples revealed that the alkaline (25mM, 50mM, and 75mM NaOH) and M-PER™ methods are efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, vaginal samples were amplified with the Promega™ PowerPlex® Fusion 6C System. Analysis of STR profiles revealed that none of the experimental methods consistently outperformed prepGEM™ across all metrics examined, including percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. However, promising results were observed in lysates treated with the M-PER™ and alkaline lysis (25mM and 50mM NaOH) reagents across all metrics, and thus these methods were subsequently tested on mixture samples. Sperm fractions of mixtures processed using prepGEM™ + alkaline lysis (1M NaOH) exhibited a mean M:F ratio of 4.14:1, which was a 6.53 ± 4.42-fold improvement over the mean ratio observed for unseparated mixture controls. Although none of the methods performed better than the control, differential lysis using alkaline lysis (25mM NaOH) + alkaline lysis (1M NaOH) outperformed the other experimental methods, exhibiting a 3.37 ± 2.30-fold improvement in the M:F ratio in the sperm fraction. These results suggest that alkaline lysis using 25mM NaOH may be an appropriate alternative cell lysis technique for non-sperm cells, but testing on the microdevice is necessary to identify any potential issues. In the future, modifications may be required to further improve these cell lysis methods and additional testing on the samples would be required to increase the confidence of our findings.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-5-2023