Document Title: APPLICATION OF PROTEINASES FOR DNA ISOLATION OF BONE SPECIMENS

One of the greatest challenges when attempting to identify victims of mass fatality incidents is the analysis of DNA from bone. The bone is more difficult to process and extract for DNA. Additionally, the initial cleaning and sampling of the bone sample is a labor-intensive and time-consuming step prior to isolating DNA. Thus, it is difficult to adapt the current method for automation. In addition, quantities of specimens recovered may be too small to properly isolate the DNA. To obtain an adequate quality and quantity of DNA templates, strategies to improve the yield of DNA isolation are needed. To address these issues, we conducted the following studies consisting two projects. Developing a simple sample processing method for DNA isolation from bone specimens. We have adapted the trypsin bone maceration method for processing bone samples prior to DNA isolation. Our results demonstrated that this method is effective for the removal of soft tissues and the outer surface of bone fragment samples. The Short Tandem Repeat (STR) analysis revealed that no adverse effect on DNA profile was detected after trypsin treatment. The yield of DNA isolated from trypsin-treated bone samples was sufficient for STR analysis. However, the DNA yield of trypsin-treated bone samples was lower than that of untreated bone samples. Our data suggest that this trypsin method is an alternative cleaning method to physical cleaning procedures, such as sanding. This method potentially has a low risk of cross-contamination between samples and diminishes safety concerns for laboratory analysts due to exposing bone powder. This method could be adapted for automated DNA isolation for human identification of bone samples, namely, from mass fatality incidents. Developing a high-yield DNA isolation method using proteinases for bone specimens. It is known that the matrix protein network plays an important role in the structure of the bone. The matrix could be digested by a number of proteinases. The hypothesis of the study is that the digestion of the matrix protein network by application of proteinases would lead to a degradation of the physical barrier surrounding the osteocytes, thus facilitating DNA extraction. Our study revealed that the clostridiopeptidase A is potent for bone degradation. The application of clostridiopeptidase A can achieve speedy and better bone degradation. The STR analysis revealed that no adverse effect on DNA profiles was detected after clostridiopeptidase A treatment. Our results demonstrated that this method improved the DNA yield of bone samples. 2 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice.