Physical and Biochemical Factors Affecting the Recovery and Analysis of DNA from Human Skeletal Remains

DESCRIPTION (provided by the applicant): There are approximately 4,400 sets of unidentified human remains recovered each year, nearly a quarter of which are not identified within the year following recovery. Obtaining genetic information through DNA testing of bone samples has become a critical element to identifying missing persons and recovered human remains. DNA is preserved within the structure of bone for vast amounts of time, surviving environmental and microbial insults, yet bone is one of the most challenging samples encountered by forensic scientists. This is due to the resilient structure of bone and the prevalence and variety of materials which co-isolate with DNA during extraction and function as inhibitors of the polymerase chain reaction (PCR). Bone-associated PCR inhibitors include native components and environmental materials, acquired as a consequence of the porous composition of bone. Quality assurance requirements governing DNA testing laboratories do not mandate direct evaluation of the product of the DNA extraction process; coupled with poor characterization of PCR inhibitors, the forensic community has not adequately demonstrated the efficiency of methods used to extract DNA from bone samples. The hypothesis to be tested is failure of PCR-based testing of DNA from skeletal remains is frequently encountered due to PCR inhibition caused by environmentally-introduced metals and inefficient extraction methods. The proposed experiments will: 1) demonstrate an approach for identifying and characterizing putative PCR inhibitors, introduced during post-mortem alteration to the mineral content and composition of bone; and, 2) assess the efficiency of current methods used for extracting DNA from bone samples, in terms of quality and quantity of the recovered template DNA. Two established methods for extracting DNA from bone will be evaluated, using standardized reference materials and controlled experimental conditions. Assessments will establish the process by which bone acquires inorganic materials from the environment, determine if the concentration of inorganic materials is sufficient to inhibit PCR-based DNA testing, and measure the efficiency of current extraction methods. Laboratory analyses will include: elemental analysis, analysis of DNA fragmentation, quantification of DNA, amplification of forensic markers, and genetic fragment analysis. Establishing a method to measure the efficiency of protocols used to extract DNA from human skeletal remains will make several important contributions toward demonstrating the significance of DNA extraction to human identification efforts. Illustrating factors that impact the success of forensic PCR-based testing will improve routine DNA extraction from bone samples, ultimately serving to increase the identification rate of human remains. This project may substantiate the development of policies that address both the methodology used to identify forensically-relevant inhibitors and the validation of forensic DNA extraction protocols. ca/ncf