Sensitivity and Species Specificity of Bisulfite Modification and Pyrosequencing Technology Used to Identify Tissue-Specific DNA Methylation Patterns in Common Biofluids Foudn in Crime Scenes

<p>In order to convict the guilty or exonerate the innocent in criminal cases, it is crucial to reconstruct the crime scene and/or determine the nature of the crime. Identifying the different biofluids found at a crime scene can help shed light on these aspects of forensic casework. Recently, DNA methylation has been used as a means of identifying biological materials, as opposed to conventional protein/enzyme based methods. DNA methylation is the addition of a methyl group to the 5' carbon on a cytosine base (C), which is directly followed by a guanine base (G) and are called CpG sites. The "p" in the CpG stands for the phosphodiester bond which connects the two bases. The study of tissue-specific DNA methylation can serve as a means of identifying the source of a biofluid. Recently, it has been observed that epigenetic DNA methylation patterns are specific for individual tissues, suggesting tissue specific differential methylation patterns. This study was undertaken to determine the sensitivity of the tests used to study the methylation pattern. Three markers, namely USP49, DDX4, and DACTl that have already been successful in differentiating sperm from other biological materials, were chosen to test the sensitivity of these technologies. The DNA quantities of 50, 10, 5, 1, 0.5, and 0.1 nanograms were used for the sensitivity study. It is also important to determine whether the markers chosen to test the sensitivity of these technologies are human specific, or if the primers will cross react with non-human biological materials. Therefore, the three previously mentioned markers were studied for their species specificity. After bisulfite modification, PCR amplification, and pyrosequencing, it was concluded that these three markers were sensitive enougn for . forensic casework in varying degrees. The USP49 and DDX4 marker produced adequate sequencing profiles from at least five nanograms of DNA, without a second round of amplification. A second round of amplification produced readable results with samples as low as 0.1 to 0.5 nanograms in DNA quantity. The DACTl marker produced interpretable profiles with quantities as low as 0.1 to 0.5 nanograms without a second round of amplification. It was also observed that the human specific primers for each marker amplified some non-human samples, when tested against cat, dog, goat, cow, chicken, macaque, chimpanzee, and Erythrobacter and Pseydomonas bacterial samples.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Masters Thesis

Discipline thesis:degree_discipline

Criminal Justice

Year dc:date.available

2013

Author dc:creator

• Shirwani, Sitara Christian

Contributors dc:contributor

• Kuppareddi Balamuragan
• Dean Bertram

• Forensic Science and Technology
• Legal Studies
• Social and Behavioral Sciences

Repository record dc:identifier

https://aquila.usm.edu/masters_theses/555

OAI identifier oai:identifier

oai:aquila.usm.edu:masters_theses-1630