Date of Award

Spring 2020

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)


Criminal Justice, Forensic Science, and Security

Committee Chair

Dr. Kuppareddi Balamurugan

Committee Chair School

Criminal Justice, Forensic Science, and Security

Committee Member 2

Dr. Dean Bertram

Committee Member 2 School

Criminal Justice, Forensic Science, and Security

Committee Member 3

Dr. Xuyang He


The identification of biological fluids is a precursor to determine if further human identification is required in a forensic setting. There are four forensically-relevant biological fluids: blood, semen, vaginal epithelial tissue, and saliva. While serological testing can identify these tissue types to some degree of accuracy, there has recently been momentum in research to use DNA methylation for tissue identification.

In the current study, five potential tissue-specific methylation markers were studied in order to identify locations in the genome that would differentiate saliva from other tissue types. Genomic DNA was extracted from each sample, followed by bisulfite modification, polymerase chain reaction amplification, and pyrosequencing. Pyrosequencing is a sequence by synthesis method that provides quantitative methylation data. The level of significance in methylation data between tissues was calculated using SPSS statistical package with a one-way ANOVA and Tukey’s posthoc parameters. Two additional studies were completed: a species-specific test and mixture study.

Five loci, cg-9652652, cg-11536474, cg-3867465, cg-10781408, and cg-10122865 along with several adjacent CpG sites were found to be hypermethylated in saliva. The methylation data of saliva was statistically significant compared to other tissues, suggesting these markers can be used to discriminate saliva from other tissue types. In the species specificity study, it was observed that the primers used in one of the assays were human specific as they did not amplify non-primate samples. A mixture study using two different tissues behaved as predicted where a reduction in the methylation percent was observed when the quantity of salivary DNA decreased.