Basic Science Methods for Clinical Researchers

Morteza Jalali, Francesca Yvonne Louise Saldanha, Mehdi Jalali

• Publisher: Academic Press
• Year: 2017
• Print ISBN:
  9780128030776
• eBook ISBN:
  9780128030783
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Basic Science Methods for Clinical Researchers addresses the specific challenges faced by clinicians without a conventional science background. The aim of the book is to introduce the reader to core experimental methods commonly used to answer questions in basic science research and to outline their relative strengths and limitations in generating conclusive data. This book will be a vital companion for clinicians undertaking laboratory-based science. It will support clinicians in the pursuit of their academic interests and in making an original contribution to their chosen field. In doing so, it will facilitate the development of tomorrow's clinician scientists and future leaders in discovery science. - Serves as a helpful guide for clinical researchers who lack a conventional science background - Organized around research themes pertaining to key biological molecules, from genes, to proteins, cells, and model organisms - Features protocols, techniques for troubleshooting common problems, and an explanation of the advantages and limitations of a technique in generating conclusive data - Appendices provide resources for practical research methodology, including legal frameworks for using stem cells and animals in the laboratory, ethical considerations, and good laboratory practice (GLP)

Chapter 1

The Polymerase Chain Reaction

PCR, qPCR, and RT-PCR

Mehdi Jalali¹, Justyna Zaborowska² and Morteza Jalali², ¹University of Liverpool, Liverpool, United Kingdom, ²University of Oxford, Oxford, United Kingdom

Abstract

The polymerase chain reaction (PCR) is a laboratory technique used for the amplification of a specific DNA fragment in a simple enzyme reaction. The basic PCR method has been modified to expand its application. Development of quantitative PCR (qPCR) has enabled detection and quantification of the target sequence in real time, while it is being synthesized. Another popular variation is reverse transcription polymerase chain reaction (RT-PCR), a technique used to detect and measure RNA. PCR technology has revolutionized the field of molecular biology and medical research. Because of its widespread use, it is important to understand the scientific principles of PCR. The aim of this chapter is to explain the concepts underlying this method and to explore the clinical usefulness and potential of this technique. The chapter also provides detailed protocols on how to undertake PCR in the laboratory, including techniques for RNA isolation, cDNA synthesis, and data analysis. A scenario in which PCR is utilized to answer a research question is also described, as well as guidance on how to troubleshoot experimental problems.

Keywords

cDNA; primer design; PCR; qPCR; RT-PCR; Taq polymerase

Chapter Outline

Introduction 2

In Principle 2

Analysis of the PCR Product 3

Phases of PCR 5

Variations of PCR Method: Quantitative PCR 5

Variations of PCR Method: Reverse Transcription PCR 8

In Practice 9

Extraction of Total RNA 9

Synthesis of cDNA by Reverse Transcription 11

Real-Time PCR Amplification 11

Data Analysis: Generating a Standard Curve 12

Relative Quantification (Pfaffl Method) 13

Applications of PCR 14

Scenario 15

Key Limitations 15

Troubleshooting 15

Little or No PCR Product 15

Efficiency of Reaction Is Too Low 15

Efficiency of Reaction Is Too High 16

Multiple Bands On Gel, Or Multiple Peaks On Melting Curve 16

No RT Control Produces a Band 16

Negative PCR Control Produces a Band 16

Conclusion 16

References 17

Suggested Further Reading 18

Glossary 18

List of Acronyms and Abbreviations 18

Objectives

Describe the scientific principles behind PCR

Provide PCR laboratory protocols

List PCR applications

Describe a typical scenario in which qRT-PCR might be used

Discuss key limitations and troubleshooting

Introduction

The polymerase chain reaction (PCR) was developed in the 1980s by Dr. Kary Mullis. The technique has been compared to a “molecular photocopier” owing to its ability to recognize a specific sequence of DNA, and rapidly and accurately synthesize a high number of copies [1]. It has revolutionized molecular biology, and in particular genetic manipulations, the diagnosis of genetic and infectious diseases, genotyping and DNA forensics. It is considered one of the greatest scientific discoveries of the 20th century. To date, a variety of spin-off techniques based on the original PCR method have been developed. Real-time PCR, also known as quantitative PCR (qPCR), combines PCR amplification and detection in a single step. Another technique known as reverse transcription polymerase chain reaction (RT-PCR) uses RNA as the nucleic acid starting template.

In Principle

PCR resembles an in vitro and elementary form of DNA replication, a physiological process used by all living cells to duplicate their genetic material prior to cell division [2]. It involves repeated cycles of heating and cooling of a reaction mixture containing DNA template, DNA polymerase, primers, and nucleotides (Table 1.1). DNA template is the DNA containing the target sequence. Primers are short chains of nucleotides which locate the specific target DNA of interest and bind to it upon cooling, through complementary base pairing. They act as a starting point for DNA polymerase to create the new complementary strand. DNA polymerase is an enzyme that synthesizes new strands of DNA complementary to the target sequence.

Table 1.1

Components of PCR and reaction guidelines for reaction optimization

Components of PCR	Guidelines
DNA TEMPLATE	Use high quality, purified DNA templates. Use 1 ng–1 μg of genomic templates per 50 μL reaction. Contaminating DNA reduces efficiency.
DNA POLYMERASE	The most commonly used...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• List of Contributors
• Foreword
• Preface
• About This Book
• Chapter 1. The Polymerase Chain Reaction: PCR, qPCR, and RT-PCR
• Chapter 2. Methods of Cloning
• Chapter 3. Whole-Mount In Situ Hybridization and a Genotyping Method on Single Xenopus Embryos
• Chapter 4. Microarrays: An Introduction and Guide to Their Use
• Chapter 5. Analysis of Human Genetic Variations Using DNA Sequencing
• Chapter 6. Western Blot
• Chapter 7. The Enzyme-Linked Immunosorbent Assay: The Application of ELISA in Clinical Research
• Chapter 8. Immunofluorescence
• Chapter 9. Cell Culture: Growing Cells as Model Systems In Vitro
• Chapter 10. Flow Cytometry
• Chapter 11. Transfection
• Chapter 12. In Vivo Animal Modeling: Drosophila
• Chapter 13. Zebrafish as a Research Organism: Danio rerio in Biomedical Research
• Chapter 14. Xenopus as a Model Organism for Biomedical Research
• Chapter 15. Basic Mouse Methods for Clinician Researchers: Harnessing the Mouse for Biomedical Research
• Appendix A. Legal Framework on the Scientific Use of Animals in Research
• Appendix B. Regulatory Frameworks for Stem Cell Research
• Appendix C. Using Multiple Experimental Methods to Address Basic Science Research Questions
• Index