Closed-Tube Barcoding of Sequence Variants (Possible Species) within the Genus Naegleria Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biology Professor Lawrence J. Wangh, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by John Deng May 2017 Copyright by John Deng © 2017 Acknowledgements I would like to thank Professor Lawrence Wangh for giving me the opportunity to pursue this project and for providing me with the financial means to make this project possible. His guidance and advice throughout this process has been invaluable. I would like to thank J. Aquiles Sanchez for his constant support and direction. I would also like to thank Nicky Sirianni for teaching me how to run experiments and Adam Osborne on the assistance and interpretation of difficult data and problems. I am grateful to Heather Schiller and to Professor Chandler Fulton for providing Naegleria samples. Finally, thank you too to all other members of the Wangh laboratory for helping to make this project successful. I have learned so much about how to be a better scientist from this process. Thank you. This research was supported in part by the Division of Science Undergraduate Research Fellowship and a Sprout grant. iii Abstract Closed-Tube Barcoding of Sequence Variants (Possible Species) Within the Naegleria Genus A thesis presented to the Department of Biology Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By John Deng This thesis provides an approach to identify and characterize the enormous diversity of Naegleria genus with Closed-Tube Barcoding. This thesis is comprised of two parts: 1) The Naegleria-Assay, which can be used to identify any species of Naegleria on earth and 2) The Fowleri-Assay, which can be used to selectively detect the “brain eating amoeba” Naegleria fowleri. In the construction of these assays, Closed-Tube Barcoding was used to generate fluorescent signatures of the CO1 gene for sequence variants of the genus Naegleria. The Naegleria-Assay can generate a unique fluorescent signature for each of the sixteen tested species of Naegleria and can even differentiate between different strains within a single species. With a few adjustments in thermal cycle and reagents, this assay can be transformed into the Fowleri-Assay, which can identify as few as ten copies of Naegleria fowleri in a background of up to 10,000 copies of DNA from other Naegleria species. The iv efficiency of this reaction is 91%. These assays indicate that a single set of probes can be used to characterize an entire diverse genus and can provide critical information about the diversity and range of the Naegleria genus as well as identify the pathogenic species Naegleria fowleri. v Table of Contents Introduction ........................................................................................................................................... 1 Traditional Methods of Species Identification are Inadequate ......................................................................... 1 Is DNA Barcoding a Solution for Rapid Identification of Species? ................................................................... 4 Benefits of DNA Barcoding? ............................................................................................................................................. 7 Problems with DNA Barcoding ....................................................................................................................................... 8 Closed-Tube Barcoding ...................................................................................................................................................... 9 Closed-Tube Barcoding of Naegleria ......................................................................................................................... 13 Materials and Methods ..................................................................................................................... 16 Final PCR Composition .................................................................................................................................................... 16 Primer Design ...................................................................................................................................................................... 17 Taq Polymerase .................................................................................................................................................................. 18 ThermaStop™ ...................................................................................................................................................................... 19 ThermaStop™ Concentration ........................................................................................................................................ 19 ThermaMark™ Concentration ...................................................................................................................................... 21 Probe Design ........................................................................................................................................................................ 21 Probe Concentration ........................................................................................................................................................ 25 Other PCR Reagents: ......................................................................................................................................................... 26 Naegleria Target DNA: ..................................................................................................................................................... 26 Target DNA Concentration ............................................................................................................................................ 28 Thermal Cycle ...................................................................................................................................................................... 28 Final PCR Composition and Setup .............................................................................................................................. 29 Results ................................................................................................................................................... 31 In Silico Tests of Naegleria Consensus Probes Against Selected Eukaryotes .......................................... 31 In Silico Test of Naegleria Consensus Probes Against the Naegleria Genus. ............................................ 34 Naegleria-Assay Fluorescent Signatures ................................................................................................................. 36 Fowleri-Assay ...................................................................................................................................................................... 49 Testing Naegleria-Assay Conditions with the Fowleri-Assay ......................................................................... 49 Increasing the Stringency of the Fowleri-Assay ................................................................................................... 50 Increasing the Copy Number of Related Naegleria Species ............................................................................. 51 Testing ThermaMark™ Concentrations .................................................................................................................... 52 Testing Different versions of ThermaMark™ ......................................................................................................... 53 Dilution series in a background of 105 copies of synthetic Naegleria gruberi DNA .............................. 55 Dilution series in a background of real Naegleria gruberi DNA .................................................................... 56 Discussion ............................................................................................................................................. 59 Naegleria Assay .................................................................................................................................................................. 59 Fowleri-Assay ...................................................................................................................................................................... 60 Challenges ............................................................................................................................................................................. 61 Future Directions ............................................................................................................................................................... 61 Conclusion ............................................................................................................................................ 63 References ............................................................................................................................................ 64 vi List of Tables Table 1: Sequence and Melting Temperatures of Fowleri-Assay and Naegleria-Assay ________ 18 Table 2: ON/OFF Probe Sequences _______________________________________________________________ 23 Table 3: List of OtHer Tested Species In-Silico ___________________________________________________