PFU DNA Polymerase: A Study of Amplification Error Rate and Subsequent Implications for High Fidelity Mutational Spectrometry by Sheila Gay Buzzee B.S. Biology, Xavier University December, 1989 Submitted to the Department of Civil and Environmental Engineering in partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY January 1994 Copyright by the Massachusetts Institute of Technology 1994. All rights reserved. Author........ .............................. Department of Cl andonmental Engineering January 26, 1994 C ertified by.....................:.-.................................,............................ .... William Thilly, Professor of Toxicology Professor of Civil and Environmental Engineering n Thesis Supervisor Accepted by ..................... ................... ................................. Professor Joseph Sussman Chairman, Department Committee on Graduate Students PFU DNA Polymerase: A Study of Amplification Error Rate and Subsequent Implications for High Fidelity Mutational Spectrometry by Sheila Gay Buzzee Submitted to the Department of Civil and Environmental Engineering on January 26, 1994, in partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering. Abstract This thesis tested the Pyrococcus furiosus (PFU) thermostabile DNA polymerase during PCR of the human mitochondrial segment encoding for tRNA glycine (bp 10,009 to 10,216). This segment is of particular interest for highly precise human mutational spectrometry studies, and the enzyme was tested to determine what amplification error rate may be anticipated in this application, and what the implications are for future human mutational studies. The enzyme was tested under conditions in which cellular DNA was amplified through 20, 40, and 60 sequential doublings so that the enzyme error rate could be determined along a wide curve. The enzyme was tested in tandem with Tli (VentTM) DNA polymerase for control purposes and error rate comparison. The enzyme was tested under standard laboratory use conditions, and tested additionally in conditions designed to fully extend incomplete extension products, hypothesized to account for a significant portion of observable background noise during mutational spectrometry using gel electrophoresis. As was previously reported by Lundberg et al [20], PFU DNA polymerase was found under standard laboratory practice to have an error rate lower than Tli (VentTM) DNA polymerase. The observed error rate is no more than 1.6 x 106 errors per base pair duplication. Furthermore this error rate was observed to be significantly reduced by additional DNA treatment to fully extend incomplete extension products. This additional treatment consisted of purification of PCR product, resuspension of the DNA, and preparation of a reaction mixture which mimics the PCR recipe but is lacking primers. This new reaction is thermocycled in the presence of PFU DNA polymerase under identical temperature conditions as PCR. This procedure allows for the complete extension of all DNA in the reaction mixture, but some DNA degradation occurs due to the high exonuclease activity of PFU. Thesis Supervisor: William G. Thilly Title: Professor of Toxicology, Professor of Civil and Environmental Engineering Acknowledgments I offer my sincere thanks and profound gratitude to Dr. John Hanekamp for his consistent support, advice, technical assistance, and unfailing friendship. Thanks also to Riitta and Jia, my wonderfully supportive and sympathetic office mates, to Hilary for keeping me company in lab at even the latest of hours, to Dr. Konstantin Khrapko and Dr. Gengxi Hu for advice, hints when I felt stuck, and sincere encouragement, and to Mark and Paula and Paulo and Aoy and all the other friendly faces for helping me keep my sense of humor no matter how tired I got. Thanks in particular to Professor Thilly for providing me with a unique environment for learning, and a forum of collaborators to discuss and experiment and create the future for ourselves. I wish you and your scientific melting pot many years of fruitful research. Thanks most importantly to my private fans and supporters- you will never know how much your encouragement has fueled me and fed me as I struggled through the long hours and (seeming) technical impossibilities. Thanks to you all, in no particular order: Mom, Dad, Sarah, Nancy, Stefan, Sarah S., Jamie, The Friday Lunch Bunch, Pat and Brian, and Ken and Jen. Contents 1 Introduction 9 2 Literature Overview 11 2.1 Mutational Spectrometry 11 2.2 Denaturing Gel Electrophoresis 11 2.2.1 Denaturing Gradient Gel Electrophoresis 11 (DGGE) 2.2.2 Constant Denaturant Capillary Electrophoresis 13 (CDCE) 2.3 Polymerase Chain Reaction (PCR) 13 2.3.1 Thermostabile Enzymes and Enzyme Fidelity 14 2.3.2 Enzyme Efficiency 15 2.4 Pyrococcusfuriosus(PFU) DNA Polymerase 16 3 Materials and Methods 17 3.1 DNA Isolation 17 3.2 Polymerase Chain Reaction 18 3.2.1 Standard PCR Procedure and Sequential 19 Amplification 3.2.2 Additional Extension Procedure 21 3.3 DNA Purification 22 3.3.1 Gel Electrophoresis 22 3.3.2 DNA Elution 22 3.4 Denaturation and Rehybridization 23 3.5 DNA Analysis 23 3.5.1 DGGE Technique 23 3.5.2 CDCE Technique 24 3.5.3 Quantification of Noise 25 4 Results 26 4.1 Sequential DNA Amplification 26 4.2 DGGE Sample Analysis 29 4.3 CDCE Sample Analysis 32 4.4 Phosphorimager Quantification of Noise 32 5 Discussion 38 5.1 Sequential Amplification 38 5.2 PFU Noise Evaluation 39 6 Conclusions 40 6.1 Direct Implications 40 6.2 Areas for Future Study 41 Bibliography 43 List of Figures 4. la PAGE visualization of first PFU amplification 28 4. lb PAGE visualization of second round of PFU amplification 28 4.1 c PAGE visualization of third round of PFU amplification 28 4. Id PAGE visualization of fourth round of PFU amplification 28 4. l1e PAGE visualization of fifth round of PFU amplification 28 4.2 DGGE of Tli VentTM versus PFU, 20 duplications 30 4.3 DGGE of PFU 20, 40, and 60 duplications 30 4.4 DGGE of terminal extension cycling and unaltered product, 31 20 duplications 4.5 CDCE of PFU amplified DNA 33 4.6 CDCE of PFU amplified multiple ss peaks 34 4.7 Phosphor scan of PFU versus Tli VentTM amplified DNA 36 4.8 Phosphor scan of terminal extension and unaltered product, 37 20 duplications List of Tables 2.1 Reported enzyme fidelities 15 3.1 PCR reaction recipe: Tli VentTM, PFU, PFU as per Lundberg et al [20] 20 4. la PCR results, first amplification 26 4. lb PCR sequential amplification, round two results 27 4. Ic PCR sequential amplification, round three results 27 4. Id PCR sequential amplification, round four results 29 4. le PCR sequential amplification, round five results 29 4.2 PFU and Tli VentTM fidelity quantification by phosphor scan 35 List of Abbreviations bp base pair(s) BSA bovine albumin serum CDCE constant denaturant capillary electrophoresis DGGE denaturing gradient gel electrophoresis dNTP deoxyribonucleoside triphosphate EtdBr ethidium bromide EtOH ethyl alcohol f enzyme fidelity HeF heteroduplex fraction hmd high melting domain lmd low melting domain MOPS 3(N-morpholino)propanesulfonic acid nt Nucleotide(s) PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PFU DNA polymerase obtained from Pyrococcusfuriosus rpm revolutions per minute ss single stranded TAE 40mM Tris-acetate, ImM EDTA (pH = 8.0 at 25 0 C) TBE 45mM Tris-borate, ImM EDTA (pH = 8.0 at 25 0 C) TEMED N,N,N',N'-tetramethylethylenediamine UV ultraviolet WT wild type Chapter 1 Introduction Mutational spectrometry is the science of determining unique and reproducible mutant patterns in DNA, and of characterizing both qualitatively and quantitatively the nature of those mutations. [1, 4 ,21, 27, 28, 29] In order to observe rare, low frequency mutants and avoid working with very large numbers of cells, it is necessary to amplify DNA for study using the polymerase chain reaction (PCR). [16, 24] Polymerase enzyme fidelity, f, is of obvious importance to mutational spectrometry- an enzyme which has poor fidelity will introduce false signals into the system. Incorrectly amplified sequences, if present in levels comparable to the expected mutant fraction, will be indistinguishable from actual mutants and may mask mutant signals altogether. This thesis tested the Pyrococcus furiosus (PFU) thermostabile DNA polymerase during PCR of the human mitochondrial segment encoding for tRNA glycine (bp 10,009 to 10,216). This multicopy segment is of particular interest for highly precise human mutational spectrometry studies, and the enzyme was tested to determine what amplification error rate may be anticipated in this application, and what the subsequent implications are for future human mutational studies. This enzyme has been reported previously to have a very high fidelity, 1.6 x 10-6 errors per base pair duplication, in a study of DNA amplification, cloning, and plating for mutant colonies. [20] Cloning is less precise than electrophoretic mutational spectroscopy and may contain internal bias in situations of inadequate sample size. Additionally the methods utilized by Lundberg et al include resin binding to remove polymerase activity and DNA eleution; both have the potential to induce mutations in DNA [10, 20]. As the enzyme fidelity reported by Lundberg is the highest of any thermostabile enzyme described to date, PFU DNA polymerase was deemed potentially very important to electrophoretic mutational