Preservation & High Throughput Methods for Human Tissue Samples in Tropical Climates; An Improved DVI Approach

As submitted by the proposer: One of the most important and immediate considerations after a mass fatality event is victim identification. Phenomena such as hurricanes, tsunamis, and acts such as the 2001 World Trade Centre attacks, wars and acts of genocide all result in large numbers of casualties. In these circumstances forensic personnel may be faced with the task of identifying hundreds or even thousands of bodily remains. The disaster victim identification (DVI) response by local and international agencies is often complicated and delayed by remote locations, harsh environmental conditions, lack of transport and a local mass disaster plan. In addition, electricity, and communication networks are often compromised, and adequate freezer facilities to house the victims do not exist. Therefore, large numbers of bodies decompose rapidly in hot and humid conditions, which lead to serious biohazard problems. However, from a DVI perspective, the DNA in those tissues is also degrading which makes genotyping more difficult with time. In these circumstances, the use of a simple field preservative to quickly halt DNA degradation and store large numbers of tissue samples at ambient temperature prior to genotyping would be a valuable tool. The research goals of this project are to develop improved DNA preservatives for tissue samples, and to optimize more rapid STR profiling methods for those samples. In-house tissue preservatives will be evaluated for how well DNA is simultaneously leeched from cadaveric muscle and skin tissue into solution, and protected from degradation when stored in hostile environmental conditions. The basic principle of salts being a highly efficient DNA preserving agent forms the argument for the solutions tested in this study. The project will focus on maximizing the quantity and quality of "free" DNA available in the surrounding preservative for rapid purification to avoid the much longer process of extracting DNA from the tissue. We will develop protocols for rapid purification, quantitation and amplification of DNA in the preservative fluid surrounding tissue samples. Strategies to quickly clean-up the lysate prior to PCR will include Microcon filtration, rapid silica columns and FTA® cards. We will also substantially (~80%) reduce the time required for amplification using the PowerPlex 18D kit (Promega). Optimization of the rapid PCR protocol will include alterations to cycle and times, polymerases, buffers and thermal cycler (Veriti, Life Technologies and Philisa, Streck). Although not a primary goal of this project, the concept of direct PCR amplification of the preservative fluid using FTA cards will also be explored. This 12 month project will be designed, directed and supervised by the Principle Investigator(PI) and Co-PI, with assistance from two graduate assistants (GA) at Sam Houston State University, Huntsville, TX. All cadaveric material will be sourced from the Southeast Texas Applied Forensic Science Facility (STAFS) based at Sam Houston State University. This facility is one of five human decomposition research facilities in the USA, and therefore offers a relatively unique opportunity to conduct the research proposed in this application. This project fulfills the requirements of the NIJ developmental goals for more cost effective, streamlined and high-throughput methods to deliver improved results for the identification of forensic evidence. Successful completion of this project will demonstrate that the novel approach of accessing DNA directly from the preservative solution surrounding a tissue sample, coupled with more rapid and direct DNA amplification can increase sample throughput during a DVI response. As a result, a significant impact can be made to address the demands for DNA preservation in rapidly decomposing remains and faster DNA identification during a mass disaster.
ca/ncf