Date of Submission
Master of Science in Forensic Science
David San Pietro
Polymerase chain reaction, Body fluids--Analysis
Body fluid detection is useful in aiding in the interpretation of events at a crime scene. The identification of body fluids can also impact the interpretation of genetic testing results through DNA. Discovering the origin of the body fluid is important to support or refute statements made involving a crime. Currently, there is not a universal confirmatory test used to accurately and specifically identify these different body fluids simultaneously. Developing a technique that can meet these criteria can be vital in circumstances where mixtures are involved or when the sample amount is limited.
DNA methylation is the addition of a methyl group onto a cytosine base in DNA. Methylation affects the functional role of transcription that in turn influences gene expression. Due to this, various tissues throughout the body display diverse methylation patterns, as they require different traits to be expressed for optimal functionality. Recent studies have found a variety of body fluids to have potentially differentiating methylation specificities. These differences could lay a foundation for a streamlined universal confirmatory test to identify multiple body fluids simultaneously.
Following the Institutional Review Board (IRB) approval, blood, saliva, vaginal fluid, and menstrual blood were collected from five female participants with informed written consent. Genomic DNA was extracted from these body fluids with the QIAamp® DNA Investigator Kit. The genomic DNA underwent a bisulfite conversion with the Qiagen Epitect® Bisulfite Kit. This kit converts unmethylated cytosine bases into uracils, allowing the methylation differences to be analyzed. Uniquely colored florescent probes were customized for previously determined methylation specific sequences for blood, saliva and vaginal fluid. The samples were analyzed with the Applied Biosystems® 7500 real time qPCR instrument with 7500 system SDS Software (v1.2.2f2) to determine cycle threshold values for each body fluid within their designated probe sequence. The CT values were also collected for each body fluid with the alternate probe sequences to compare methylation expression differences, as well as any possible cross reactivity.
Pascu, Mandy A., "Forensic Body Fluid Identification with the Epitect® Bisulfite Kit and the Reliablity of DNA Methylation with Real Time qPCR" (2019). Master's Theses. 114.
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