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A Technical Guide PCR TECHNOLOGIES • PCR • RT-PCR • Qpcr • RT-Qpcr • Dpcr

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A Technical Guide PCR TECHNOLOGIES • PCR • RT-PCR • Qpcr • RT-Qpcr • Dpcr A Technical Guide PCR TECHNOLOGIES • PCR • RT-PCR • qPCR • RT-qPCR • dPCR Foreword The introduction of the Polymerase Chain Reaction (PCR) This guide contains the material presented during technical has revolutionized identification and measurement of DNA workshops and therefore covers the questions that are often sequences and RNA when used in conjunction with reverse raised, and answers the questions we encounter on a day- transcription. The technique has become ubiquitous across to-day basis when offering technical support. This guide is all fields of life science and has been adapted to provide presented as a complete solution to those working with PCR- quantitative analysis (qPCR) and more recently copy number based techniques. determinations (dPCR). All of these PCR-based techniques are conceptually and practically simple to implement and, with Editor appropriate controls, yield highly informative data. However, Tania Nolan, Ph.D. the vast quantity of information flooding both the peer reviewed and popular literature, is daunting to those wishing Contributors to embark on a PCR experiment or adopt a new technique. Anders Bergkvist, Ph.D. The situation is made more difficult by conflicting advice Carol Carvallo, M.S. and recommendations. During the preparation of the MIQE Peter H. Chereson, Ph.D. recommendations1, a publication dedicated to clarifying the Laura Daley, Ph.D. information required for a qPCR experiment to be evaluated, Ashley Heath, Ph.D. and the subsequent evaluation of peer reviewed papers Suzanne Hibbs, M.S. relying on qPCR data2, it became clear that there was a Stacey Hoge, B.S. desperate need for detailed guidance on the essentials of how Elena Jouravlena, Ph.D. to carry out PCR techniques and why particular practices may Carol Kreader, Ph.D. be recommended under specific circumstances. This guide Mudassir Mohammed, Ph.D. has been written to address that need. Ernie Mueller, Ph.D. While there are many excellent textbooks and guides available, Genova Richardson, M.S. this is a unique offering in that it provides detailed technical Tom Russell, Ph.D. explanations and protocols for PCR and RT-PCR techniques, Brian Ward, Ph.D. including specific information relevant to qPCR and digital Scott A. Weber, B.S. PCR. Many popular myths have been addressed and a detailed Marina Wiklander, Ph.D. troubleshooting guide provided that includes training on the References appropriate use of controls and how to apply this information 1. Bustin, S.A., Benes, V., Garson J.A., et al. The MIQE guidelines: minimum to any experiment. It is clear from the requests for support that information for publication of quantitative real-time PCR experiments. we receive, that data analysis is a bottleneck in the scientific Clin Chem 2009, 55, 611-622. process and so this is also addressed in a separate chapter. 2. Bustin, S.A., Benes, V., Garson J., et al. The need for transparency and good practices in the qPCR literature. Nat Methods 2013, 10, 1063-1067. Table of Contents Table of Contents Chapter 1: Introduction and Historical Timelines Chapter 8: Reverse Transcription Introduction 3 Introduction 53 Historical Timelines 3 Reverse Transcription Linearity 53 Reverse Transcription Priming 54 Chapter 2: Polymerase Chain Reaction Reverse Transcription Efficiency 55 Cycling Procedure 7 PCR Assay Components 9 Chapter 9: Assay Optimization and Validation Instruments 9 Introduction 59 Application-specific PCR Modifications 10 Validating Primer Design 59 Optimizing Probe Concentration 63 Chapter 3: Quantitative PCR Optimizing Reaction Components and Multiplex Assays 63 Basic Principles 11 Optimizing Mg2+ Concentration 63 Quantification and Analysis of gDNA Targets 14 Probe Fluorophore and Quencher Selection 63 Quantification and Analysis of mRNA Transcripts 14 Guidelines for Optimization of Quantitative Reverse Quantification and Analysis of ncRNA Transcripts 14 Transcription PCR (RT-qPCR) 64 qPCR Requirements 15 Assay Evaluation 65 The MIQE Guidelines 16 Chapter 10: Data Analysis Chapter 4: Digital PCR PCR/qPCR Qualitative Analysis 69 Instruments 20 qPCR Data Analysis 69 Instrument Limitations 21 Deriving Accurate Cq Values 69 dPCR Applications 22 Setting the Threshold 70 Digital PCR MIQE 23 qPCR Quantification Strategies 73 Chapter 5: Quantitative PCR and Digital PCR Detection Methods Standard Curve Quantification 73 Relative/Comparative Quantification 73 Introduction 25 Normalization 74 dsDNA-Binding Dyes 25 Reference Gene Selection 75 Probes 26 Analysis of Reference Gene Stability 76 Quenchers 28 Alternative Normalization Methods 78 Nucleic Acid Analogs 29 Statistical Analysis and Data Visualization 80 Summary 30 Statistical Tests 80 Chapter 6: PCR/qPCR/dPCR Assay Design Visualization Techniques for Univariate Analysis 81 Hierarchical Clustering 82 Introduction 31 Principal Component Analysis 83 Amplicon Selection 31 Methylation-specific Assays 33 Chapter 11: Troubleshooting Primer Design 33 Developing a Troubleshooting Protocol 85 Probe Design 36 Troubleshooting Examples Demonstrating the Use of the Oligo Synthesis and Handling 38 Diagnostic Tools 88 Oligonucleotide Purification 38 Troubleshooting Case Studies 98 Oligonucleotide Preparation 39 Summary—A Troubleshooting Protocol 99 Chapter 7: Sample Purification and Quality Assessment Chapter 12: Regulatory Agencies and Conclusions Introduction 41 Regulatory Landscape Overview 101 Nucleic Acid Purification 41 Assay Validation 102 Chaotropic Agents 41 Approved Tests 103 Extraction of Genomic DNA 41 Conclusions 103 Purification of Total RNA 42 Next-generation Sequencing 105 Nucleic Acid Sample Quality Assessment 43 Additional Technical Support 105 Gel Electrophoresis and Capillary Nucleic Acid Quality Analysis Systems 45 Contamination 48 Inhibition 49 An Integrated Example: Analysis of RNA Sample Quality 49 PCR Technologies: A Technical Guide 1 Appendix A: Protocols Optimization of qPCR Conditions Introduction 107 Protocol 13: Primer Concentration Optimization 137 Protocol 1: OligoArchitect Assay Design 110 Protocol 14: Primer Optimization Using Temperature Gradient 139 Protocol 15: qPCR Reference Gene Selection 141 End-point PCR Protocols Protocol 16: qPCR Efficiency Determination 143 Protocol 2: Antibody–Enzyme Mediated Hot Start PCR 116 Protocol 17: qPCR Gene Expression/Copy Number Analysis Using Protocol 3: dNTP-Mediated Hot Start PCR 118 SYBR Green I Dye Detection 145 Quantitative PCR Protocols Protocol 18: qPCR Gene Expression/Copy Number/SNP Analysis Using Probe Detection 147 Protocol 4: SYBR Green I Dye Quantitative PCR 121 Protocol References 149 Protocol 5: qPCR Using a Single Detection Probe 124 Protocol 6: Multiplex qPCR 126 Appendix B: Additional Resources Protocol 7: dPCR 128 Books, Seminars and Tools 151 Protocol 8: The SPUD Assay for Detection of Assay Inhibitors 130 Products and Support 154 Protocol 9: The 3’/5’ Assay for Analysis of RNA Integrity 132 Design Your Probe Online with OligoArchitect 158 Reverse Transcription Trademarks Protocol 10: Standard Reverse Transcription Protocol (Two-step) 134 Protocol 11: Reverse Transcription Protocol (One-step SYBR Green I Dye Detection) 135 Protocol 12: Reverse Transcription Protocol (One-step Probe Detection) 136 2 sigma.com Introduction and Historical Timelines Chapter 1: Introduction and Historical Timelines While this passage appears to be a clear description of Introduction the process that is now recognized as the PCR, it could The Polymerase Chain Reaction (PCR) is used in all areas of not be verified experimentally at the time because the biological science research, including the clinical, forensic and target sequences required for primer design were not diagnostic fields and the widespread adoption of the PCR readily available, nor was there a convenient method for technique has revolutionized life science research. manufacturing the synthetic PCR primer oligonucleotides. Many improvements have been made to the basic technique Originally, when considering the process of DNA amplification, and many adaptations have evolved, including reverse Kary Mullis, et al.,2 had assumed that when primers were added transcription PCR (RT-PCR), quantitative real-time PCR (qPCR), to denatured DNA, they would be extended, the extension reverse transcription qPCR (RT-qPCR) and digital PCR (dPCR). products would become unwound from their templates, be This guide is designed for scientists with a background in primed again and then the process of extension repeated. molecular biology and for scientists in other fields who are However, this was not the case and the DNA had to be heated interested in learning more about PCR-based techniques. Basic almost to boiling after each round of synthesis to denature the principles are presented and supplemented with practical newly formed, double-stranded DNA. The Klenow fragment of examples and theoretical concepts. DNA polymerase I that was being used for synthesis was then inactivated by the high temperature and so more enzyme was To illustrate the theoretical concepts, protocols are provided required at the start of each cycle, as predicted by Kleppe1. that can be used to analyze sample quality, optimize assay A critical development that led to the universal adoption of conditions, determine assay efficiency and RT linearity. These the PCR technique was the concept of using a thermal stable protocols can be easily adapted for all research applications. DNA polymerase that could tolerate the high temperature of the repeated denaturation steps. The Klenow fragment Historical Timelines of DNA polymerase
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