University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Biochemistry Biochemistry, Department of 5-2012 Developing a High Throughput Protocol for Using Soil Molecular Biology as Trace Evidence Sabreena A. Larson University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/biochemdiss Part of the Biochemistry, Biophysics, and Structural Biology Commons Larson, Sabreena A., "Developing a High Throughput Protocol for Using Soil Molecular Biology as Trace Evidence" (2012). Theses and Dissertations in Biochemistry. 9. https://digitalcommons.unl.edu/biochemdiss/9 This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Biochemistry by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. DEVELOPING A HIGH THROUGHPUT PROTOCOL FOR USING SOIL MOLECULAR BIOLOGY AS TRACE EVIDENCE By Sabreena Larson A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Biochemistry Under the Supervision of Professor Cheryl P. Bailey Lincoln, Nebraska May 2012 DEVELOPING A HIGH THROUGHPUT PROTOCOL FOR USING SOIL MOLECULAR BIOLOGY AS TRACE EVIDENCE Sabreena Larson, M.S. University of Nebraska, 2012 Adviser: Cheryl P. Bailey The use of soil as trace evidence has changed significantly with the addition of new techniques. These techniques include using the biochemical molecules from soil microbial communities to make a fingerprint of the specific soil. This research examines the changes to the microbial community profile that take place during storage of a soil sample. To observe such changes both the DNA and fatty acid profiles will be examined. The DNA profiles were made with capillary electrophoresis-single stranded conformation polymorphism (CE-SSCP). After statistical analysis using Bray-Curtis distances and ANOSIM (analysis of similarity) it was shown that storage of soil does not have a significant impact on the microbial community profile. However, when samples were compared across soil collection sites significant differences were seen. This illustrates that different soils respond differently to storage treatments. The fatty acid profiles were analyzed as fatty acid methyl esters (FAMEs) using gas chromatography. Data were analyzed using canonical correlation analysis, squared Mahalanobis distance, and repeated measures. The results show that -80˚C is the best iii way to store soils to preserve the integrity of the microbial community FAME profile, followed by -20˚C. It was also demonstrated that when using fatty acids to examine the change within the soil at the collection site there is generally not a significant difference between the soil collected over a two week period. When the two methods are compared FAME is a more sensitive method to minute changes within the microbial community. With the data from these two methods, using soil microbial community profiling is closer to becoming a viable option for forensic science. iv Dedication I dedicate this thesis and all the hard work put into it over the last two years to my parents, Randy and Ruth Bathke. To my father, for always encouraging me to pursue my passion of science. To my mother, for often reminding me to enjoy what I do and the people around me for one day it will be gone. Thank you both for your support and encouragement. Author’s Acknowledgments I would like to thank Dr. Cheryl Bailey for accepting me into her lab and her support and encouragement throughout this process. I would also like to thank Dr. David Carter for helping to keep the projects moving forward and always lending an ear. Both of you were instrumental in my success of this project. Thank you. To my other committee members, Dr. Rhae Drijber, Dr. Don Weeks, and Dr. Ashley Hall, thank you for helping me to trouble shoot the many issues that arouse. To Dr. Rhae Drijber for your invaluable knowledge in fatty acids and soil microbial communities. To Dr. Don Weeks for helping to guide me through the process of a master’s degree and your extensive knowledge in biochemistry. Dr. Ashley Hall thank you for your support and help with the genetic analyzer. This work would not be possible without the help of Niraj Patel and Victoria Freeman. Thank you for all your hard work in completing the many tasks that were given to you. I appreciate your cooperation and communication to ensure the project was completed. To the entire Bailey lab for good teamwork and support throughout the project. To my family, my loving husband, Blake Larson, who supported during this process, of obtaining this degree and to my adorable son, Matthew Larson, who reminded me to keep things in perspective. I thank you for your support over the past two years. Without it this would not have been possible. v Table of Contents INTRODUCTION……………………………………………………......................…...ii DEDICATION…………………………………………………………………………...iv ACKNOWLEDGEMENTS………………….…………………………….……….……iv TABLE OF CONTENTS………………………………………………………….……..v LIST OF FIUGRES……………………………………………………………….……..vi LIST OF TABLES………………………………………………………………….……vi CHAPTER 1: Introduction and Literature Review………………………………………1 CHAPTER 2: Changes in DNA Profiles of Soil Microbial Communities Due to Storage and Handling…………………………………………………………….……….……...23 Abstract………………………………………………………………………………….24 Introduction……………………………………………………………………………...24 Methods………………………………………………………………………………….25 Results……………………………………………………………………………………29 Discussion………………………………………………………………………………..31 References………………………………………………………………………………..34 Figures……………………………………………………………………………………36 CHAPTER 3: Changes in Fatty Acid Profiles of Soil Microbial Communities Due to Storage and Handling…………………………………………………………………….46 Abstract…………………………………………………………………………………..47 Introduction………………………………………………………………………………48 Methods…………………………………………………………………………………..49 Results……………………………………………………………………………………52 Discussion………………………………………………………………….…………….55 References………………………………………………………………….…………….59 Figures……………………………………………………………………..……………..61 CHAPTER 4: Synthesis and Conclusion ……………….………………………………78 APPENDIX A: Soil Collection Sites …………………………...………………………81 APPENDIX B: DNA ANOSIMs Results………………………………………………88 vi List of Figures Chapter1 Figure1. USDA soil texture chart…………………………………………………….…22 Chapter2 Figure1. Bray-Curtis similarity index untransformed fresh soil samples from four sites by season and forward and reverse primer……….……………………………………...37 Chapter3 Figure1. Discriminant/canonical correlation analysis of storage treatments over all seasons and collection sites………………………………….…………………………..61 Figure2. Discriminant/canonical correlation analysis of soil site by storage treatment...63 Figure3. Discriminant/canonical correlation analysis of season by storage treatment.....65 Figure4. Discriminant/canonical correlation analysis of overall treatment and by individual seasons……………………………………………………………………….68 Appendix A Figure1. Soil collection calendar for September 2010……………………………….…84 Figure2. Soil collection calendar for November 2010……………………………….…85 Figure3. Soil collection calendar for July / August 2011…………………………….…86 Figure4. Aerial view of soil collection sites………………………………………..…...87 List of Tables Chapter2 Table1. Soil characteristics from the four soil collection sites………………………...36 Table2. Analysis of similarity P values for all soils combined examining significant differences of storage treatments……………………………………………………....43 Table3. Analysis of similarity P values for forward primers of individual soils examining significant differences of storage treatments…………………………………………...44 Table4. Analysis of similarity P values for reverse primers of individual soils examining significant differences of storage treatments……………………………………………45 Chapter3 Table1. Chemical and physical characteristics of the four soil collection sites………36 Table2. Squared Mahalanobis distances of location by storage treatment…………...64 Table3. Squared Mahalanobis distances of season by storage treatment……………67 Table4. Squared Mahalanobis distances of storage treatment by seasons…………,,,76 Table5. Repeated measures of soil microbial biomass by storage treatments ………77 Appendix A Table1. Complete physicochemical characteristics for all soil sites…………………..82 Table2. Examination of specific elements within all four soil sites…………………...83 Table3. Soil textures in percent sand, silt, and clay…………………………………....83 Appendix B vii Table1. Analysis of similarity R values for all soils combined of storage treatments compared to fresh samples for three seasons…………………………………………..89 Table2. Analysis of similarity R values of forward primers: storage treatment compared to fresh from four soil sites over three seasons….……………………………………..90 Table3. Analysis of similarity R values of reverse primers: storage treatment compared to fresh from four soil sites over three seasons…….…………………………………..91 1 Chapter 1 Introduction and Literature Review 2 Chapter 1 I. Introduction The Project The growing interest in using the soil microbial community to fingerprint a soil sample for forensic science purposes has opened the door to further investigate the details of this potential trace evidence. Theoretically, the soil microbial community can be used to link a suspect or victim to a crime scene or confirm / contradict an alibi by comparing the