Date of Graduation

2015

Document Type

Thesis

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Clifton Bishop

Committee Co-Chair

Jennifer Hawkins

Committee Member

Tina Moroose

Abstract

There are several methods that are currently used to estimate a postmortem interval (PMI), including physiological, biochemical, entomological, and archeological approaches. However, these methods are not effective in estimating PMI's extending from several weeks to months. The study presented here was performed to create an independent, objective biological method to estimate PMI by analyzing the degradation of 18S ribosomal RNA (rRNA) from porcine and human dental pulp. If successful, this method would overlap and extend the current PMI estimation methods. With a concept similar to that of radioactive carbon-14 dating, this method analyzed two different sized segments -- one large, labile segment and one small, stable segment - of the same rRNA strand using real-time reverse transcription polymerase chain reaction (RT-PCR). Since the larger segment acts as a larger target for degradation forces, it is expected that the larger PCR amplicons will disappear more quickly than the smaller amplicons, and it is the difference in the quantities of each amplicon that is expected to help estimate a PMI. A multiplex reaction with a designated fluorescent marker for each larger and smaller segment was used to examine both segments of the RNA within a single sample. Teeth and weather data were collected weekly. Once the dental pulp was isolated, the RNA was extracted and converted to complimentary DNA (cDNA) using reverse transcription PCR. Once in the cDNA form, the samples were analyzed using real-time PCR. Colorimetric data was also collected by taking a picture of the pulp sample, and using Adobe Photoshop to quantify the color content of each photograph. As PMI increased, the tooth pulp experienced some color and morphological changes, as well as degradation of the rRNA target which allowed for a relative estimate of time since death with porcine samples. Using the weather data collected, accumulated degree days (ADD) for each sample were calculated and helped refine the PMI estimate. Unfortunately, the human samples were not as successful, possibly due to the age of the specimens studied. One study of an 18-year old specimen produced promising results which were similar to those obtained with the porcine samples. However, due to some resource restraints, further analyses could not be performed on younger human specimens in this particular study, though it was evident that age of the human specimens had a robust effect on the method outcome. Still, this method can be of great significance to the forensic field, especially since PMI estimates get more complicated as decomposition progresses. This method can also fill the time gap between the maximum amount of time insects can be used to estimate a PMI and the minimum amount of time archeological approaches can be applied. Determining an accurate PMI estimate is crucial to recreating a possible homicide event and will be extremely beneficial to forensic investigations. Using a technique such as the one herein may assist investigations that may not have been able to be solved before without an accurate PMI estimate.

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