Development of a Quantitative PCR (qPCR) Based Method for Studying Temporal DNA Degradation in Waterlogged Bone
Directed Research Project
Human activities are often centered around the presence of water, thus it is not surprising that there are many water-related human deaths. Accumulated degree days (ADD), and other aquatic variables may affect DNA retrieval from waterlogged bone. Calcium and collagen in bone can inhibit the PCR necessary to produce an STR profile; the current solution is a time-consuming organic extraction. While there are examples of research on DNA degradation in terrestrial bone over time, there has been little work done on submerged bone samples and they are usually limited to case studies. The major aim of this study was to measure host DNA quality and quantity in porcine waterlogged bones over time/ADD. It was accomplished by 1) attempting to optimize qPCR protocol for host DNA quantification and degradation index (DI) estimation, 2) determining the best extraction method (ChargeSwitch® gDNA Plant Kit v organic phenol-chloroform), and best bone type (between rib or scapula) for host DNA recovery in freshwater environment using a qPCR based method, and 3) identifying the variance of host DNA recovery in different bone types and water bodies. A SYBR based quantitative PCR protocol was developed for quantification host DNA using two target DNA loci (larger fragment target: 274-314bp and small fragment target: 93-127bp). The protocol was highly effective with the chosen STR primers, with the organic method obtaining the highest quantity with the lake samples, specifically in the ribs. There was evidence of a significant difference in degradation index over time, and the scapulae having the higher degradation index between bone samples. Individually, there was no significant difference in bone, method or location when it came to DNA quantity; combined interactions were required to find significance. Overall, scientists now have the opportunity to implement a more streamlined, efficient workflow from sample prep to profile development, which is pivotal in identification matters where time and resources are of the essence.
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VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
Biology Commons, Criminology and Criminal Justice Commons, Forensic Science and Technology Commons, Molecular Biology Commons