Date of Submission
Master of Science in Forensic Science
Brooke W. Kammrath, Ph.D.
Raman Microspectroscopy, Screening Tool, DNA Recovery, Human Skeletal Remains
Spectrum Analysis, Raman, Hyperspectral Imaging, DNA, Body Remains
Raman spectroscopy, Hyperspectral imaging, DNA--Analysis, Dead
Numerous challenges exist with forensic genetic testing of human skeletal remains due to diagenesis patterns in bone microstructure, DNA degradation, and the presence of PCR inhibitors. Diagenesis is the microscopic breakdown of the bone matrix, which consists primarily of mineralized calcium hydroxyapatite and collagen. The process of diagenesis occurs in a heterogeneous, non-uniform manner along the diaphysis of a long bone, and determining the region with the most intact bone microstructure is not possible with the naked eye. Therefore, taking cuttings from the diaphysis for DNA testing is somewhat of a “blind” process, and decades of research and casework have demonstrated that differences in DNA recovery do exist between cuttings along the shaft of the same long bone. An additional consideration is that forensic genetic testing of bones is a time-consuming and labor-intensive process. Development of an effective screening method to determine the optimal sampling site(s) on the diaphysis could reduce time, labor, costs, and the degree of destructive sampling necessary to obtain a DNA profile. This approach could help maximize DNA recovery and improve success rates in unidentified human remains (UHR) investigations.
Raman spectroscopy was evaluated as a reliable screening tool to obtain information about bone microstructure and stage of diagenesis which, according to previous research, often correlates to the quantity and quality of endogenous DNA within that region of bone. In the first phase of this research, Raman spectroscopy was evaluated for its effects on known quantities of human DNA extracted from buccal swabs. This step was implemented to determine if exposure to the Raman laser would damage endogenous DNA, which would preclude the use of spectroscopy in genetic casework involving human skeletal remains. Initial results suggested that possible contamination and/or heat evaporation occurred, which would then skew calculations for ii determining the amount of DNA (ng) present in the sample after Raman exposure. Thus, in order to eliminate these possibilities, additional samples were taken and the total volume (μl) was carefully measured and calculated via incremental pipetting until all liquid has been recovered from the cap. These results, now precisely and accurately measured, demonstrated that DNA was damaged (both quantitatively and qualitatively) after Raman exposure.
Additionally, a fresh non-human (mammal) bone was scanned to serve as a reference for high-quality (non-degraded) bone microstructure. In the second phase of this research, Raman spectroscopy was used to scan various pre-marked sections of the diaphysis of long bones from two sets of human skeletal remains with varying post-mortem intervals (i.e., 5-year PMI, 50-year PMI). Compositional analysis of each scanned section provided information regarding degree of diagenesis within the bone microstructure. The parameters determined through experimentation that were necessary to acquire a consistent, good spectra of the bone actually burned/charred the bone. For this reason, current data does not support the use of Raman spectroscopy as a prescreening tool for bone prior to DNA testing because it is not only destructive to DNA, but also to bone microstructure. However, this research provided further support for the recommendation to sample from more than one area/region of the diaphysis by demonstrating the non-uniform, heterogeneous process of diagenesis.
Smith, Kathleen M., "Evaluation of Raman Microspectroscopy and Hyperspectral Imaging as a Screening Tool to Determine Optimal Sampling Sites for DNA Recovery from Human Skeletal Remains" (2023). Master's Theses. 192.