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Molecular Thermodynamics of the Stability of Natural, Sugar MOLECULAR THERMODYNAMICS OF THE STABILITY OF NATURAL, SUGAR AND BASE-MODIFIED DNA DUPLEXES AND ITS APPLICATION TO THE DESIGN OF PROBES AND PRIMERS FOR SENSITIVE DETECTION OF SOMATIC POINT MUTATIONS by Curtis Hughesman B.A.Sc., The University of Calgary, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2012 © Curtis Hughesman, 2012 Abstract Cancer is characterized as a genetic disease associated with acquired somatic mutations, a majority of which consist of only a single base change and are commonly referred to as somatic point mutations (SPM). Real-time quantitative polymerase-chain reaction (qPCR) techniques using allele specific (AS) probes or primers are widely used in genotyping assays to detect commonly known single nucleotide polymorphisms (SNP), and also have the potential to detect SPMs, provided the required analytical sensitivity and specificity can be realized. One strategy to establish the necessary performance is to introduce nucleotide analogs such as Locked Nucleic Acids (LNAs) into AS probes or primers; however the successful design requires a fundamental understanding of both the thermodynamics and kinetics of LNA-DNA heteroduplexes. Melting thermodynamic studies of DNA duplexes and LNA-DNA heteroduplexes were therefore carried out using both ultraviolet (UV) spectroscopy and differential scanning calorimetry (DSC) to quantify the o o thermodynamics (ΔH , ΔS , ΔCp and Tm) associated with the helix-to-coil transition. Data collected on DNA duplexes and DNA-LNA heteroduplexes were used to introduce improvements in the “unified” nearest-neighbor model, and for the development of a new model, referred to as the Single Base Thermodynamic (SBT) model that accurately predicts the Tm for the melting of LNA-DNA heteroduplexes. The SBT model was extended and applied to PCR conditions to design LNA-bearing AS probes for qPCR assays to detect the clinically important SPMs KIT c.1799t>a (D816V) and JAK2 c.1849g>t (V617F), and were found to significantly outperform standard AS probes containing only DNA. The interaction of Taq polymerase with heteroduplexes formed between an LNA-bearing primer and a target template were also studied and results ii used to generate general rules for designing LNA-bearing AS primers capable of unequivocal detection of a rare mutant allele bearing a SPM. The method was then extended to allow qPCR detection by Plexor™ technology and applied to create an AS primer directed against the JAK2 V617F SPM that can detect one mutation in a background of more than 100,000 copies of the wild-type allele and which is now used by the Cancer Genetics Laboratory of the British Columbia Cancer Agency (BCCA) to analyze patient samples. iii Preface A version of Chapter 1 has been submitted for publication to "Biochemical Engineering Thermodynamics", in press, von Stocker U, et al. (Eds.), EPFL Press (2012). The manuscript was written through collaboration between Dr. Charles Haynes and me. A version of Chapter 2 has been published. Hughesman, C.B., Turner, R.F. and Haynes, C. (2011) Correcting for Heat Capacity and 5'-TA Type Terminal Nearest Neighbors Improves Prediction of DNA Melting Temperatures Using Nearest-Neighbor Thermodynamic Models. Biochemistry, 50, 2642-2649. I performed all of the experiments and wrote most of the manuscript. Dr. Charles Haynes provided guidance on the research. Dr. Charles Haynes and Dr. Robin Turner reviewed and edited the manuscript. A version of Chapter 3 has been published. Hughesman, C.B., Turner, R.F.B. and Haynes, C.A. (2011) Role of the Heat Capacity Change in Understanding and Modeling Melting Thermodynamics of Complementary Duplexes Containing Standard and Nucleobase- Modified LNA. Biochemistry, 50, 5354-5368. I performed or directly supervised all of the experiments and wrote most of the manuscripts. Dr. Charles Haynes provided guidance on the research. Dr. Charles Haynes and Dr. Robin Turner reviewed and edited the manuscript. A version of Chapter 5 is being prepared for submission as a publication. Experiments on the LNA-bearing primers directed at the BCL2 plasmids were performed by Colin Olsen and me. Kelly McNeil generated the JAK2 plasmids and DNA from patients for testing. All experiments involving the AS primers were performed by myself. Kelly McNeil, Sean iv Young, Dr. Aly Karsan and Dr. Charles Haynes provided guidance on the research and experiments. Approval by UBC’s Research Ethics Board was obtained; blind testing of anonymous patient samples previously acquired and stored at the BC Cancer Agency using assays developed in this work was conducted under UBC’s research ethics certificate H08- 01035. All testing was conducted for research purposes only and no knowledge of patient identity or medical history was known or transferred. v Table of Contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of Contents ................................................................................................................... vi List of Tables .......................................................................................................................... ix List of Figures ........................................................................................................................ xii Nomenclature ........................................................................................................................ xv Acknowledgements ............................................................................................................ xviii Dedication .............................................................................................................................. xx Chapter 1: Introduction ........................................................................................................ 1 1.1 Thesis Overview ....................................................................................................... 1 1.2 Background ............................................................................................................... 7 1.2.1 Methods for measuring duplex DNA melting thermodynamics ..................... 12 1.2.1.1 UV absorption spectroscopy ....................................................................... 13 1.2.1.2 Calorimetry ................................................................................................. 20 1.2.2 Thermodynamic models used to predict DNA duplex stability...................... 24 1.2.3 Locked Nucleic Acids (LNAs) ....................................................................... 30 1.2.3.1 Chemistry and properties ............................................................................ 31 1.2.3.2 Predicting the stability of LNA-DNA heteroduplexes ................................ 32 1.2.4 PCR based methods for detection and quantification of a SPM ..................... 34 1.2.4.1 LNA containing AS probes ......................................................................... 38 1.2.4.2 LNA containing AS primers ....................................................................... 39 1.2.5 Clinically significant somatic point mutations ............................................... 41 1.2.5.1 JAK2 V617F................................................................................................ 41 1.2.5.2 KIT D816V.................................................................................................. 42 1.2.5.3 BRAF V600E .............................................................................................. 42 1.3 Thesis Objectives and Content Overview ............................................................... 43 Chapter 2: Correcting for Heat Capacity and 5'-ta Type Terminal Nearest Neighbors Improves Prediction of DNA Melting Temperatures Using Nearest-Neighbor Thermodynamic Models ....................................................................................................... 46 2.1 Materials and Methods ............................................................................................ 47 2.1.1 DNA synthesis and purification ...................................................................... 47 2.1.2 Differential scanning calorimetry ................................................................... 47 2.1.3 Regression of melting thermodynamics data .................................................. 48 2.1.4 Error analysis .................................................................................................. 48 2.2 Results and Discussion ........................................................................................... 49 2.2.1 Introduction of ΔCp into the unified NNT model improves Tm predictions ... 51 bp 2.2.2 Regressed ΔCp and Tref values are supported by DSC data .......................... 55 2.2.3 Duplexes terminating in a 5’-ta have statistically significant Tm(error) ............ 59 2.2.4 Correcting Tm predictions for duplexes containing 5’-ta type termini............ 63 2.3 Conclusions ............................................................................................................. 65 Chapter 3: The Role of the Heat
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