PCR Troubleshooting and Optimization: The Essential Guide | Book
"an essential book ... a valuable tool to all those interested in PCR" (Doodys)
"an essential guide" Aus. J. Med. Sci.
Caister Academic Press
Suzanne Kennedy and Nick Oswald
MO BIO Laboratories, Inc., Carlsbad, CA 92010, USA and BitesizeBio, Edinburgh, UK (respectively)
viii + 236
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The polymerase chain reaction (PCR) is a fundamental tool in scientific research and clinical testing. Real-time PCR, combining both amplification and detection in one instrument, is a rapid and accurate method for nucleic acid detection and quantification. Although PCR is a very powerful technique, the results achieved are valid only if the appropriate controls have been employed. In addition, proper optimization of PCR conditions is required for the generation of specific, repeatable, reproducible and sensitive data.
This book discusses the strategies for preparing effective controls and standards for PCR, when they should be employed and how to interpret the information they provide. It highlights the significance of optimization for efficiency, precision and sensitivity of PCR methodology and provides essential guidance on how to troubleshoot inefficient reactions. Experts in PCR describe design and optimization techniques, discuss the use of appropriate controls, explain the significance of standard curves and explore the principles and strategies required for effective troubleshooting. Authors highlight the importance of sample preparation and quality, primer design, controlling inhibitors, avoiding amplicon and environmental contamination, optimizing reagent quality and concentration, and modifying the thermal cycling protocol for optimal sensitivity and specificity. In addition, specific chapters discuss the history of PCR, the choice of instrumentation, the applications of PCR in metagenomics, high resolution melting analysis, the MIQE guidelines, and PCR at the microliter scale.
The strategies, tips and advice contained in this concise volume enable the scientist to optimize and effectively troubleshoot a wide range of techniques including PCR, reverse transcriptase PCR, real-time PCR and quantitative PCR. An essential book for anyone using PCR technology.
"a comprehensive selection of the most recently developed applications ... an essential book for investigators using PCR technology ... a well-balanced book ... It also provides a number of updated strategies for investigators interested in incorporating this technique in their research. The presentation is straightforward and is based on proven examples. The book should prove to be a valuable tool to all those interested in PCR technology." from Doodys
"The comprehensive and comprehendible content indeed qualifies the text as an essential guide to the development, optimization and toubleshooting of PCR assays." from Aus. J. Med. Sci. (2011) 32: 68
Table of contents
1. Magic in Solution: An Introduction and Brief History of PCR
Carl T. Wittwer and Jared S. Farrar
The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. The origins of PCR and its early evolution are described, including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. Early real-time instruments are compared. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.
2. Difficult Templates and Inhibitors of PCR
Jack M. Gallup
One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay. To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, this chapter briefly explores some of the current strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. It also discusses strategies to amplify difficult templates and optimize reverse transcription reactions.
3. Significance of Controls and Standard Curves in PCR
Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab
Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. This chapter discusses the strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. It also highlights the significance of standard curves for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It also provides guidance on how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.
4. Obtaining Maximum PCR Sensitivity and Specificity
Cameron N. Gundry and Matthew D. Poulson
PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. This chapter will address all of these factors to aid the investigator in designing high quality PCR assays.
5. RT-PCR Optimization Strategies
Martina Reiter and Michael W. Pfaffl
PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. In this chapter the optimization of the whole RT-PCR workflow will be discussed and recommendations to reduce experimental variance and produce more reproducible and reliable results are put forward.
6. Real-Time PCR Instrumentation: An Instrument Selection Guide
Sandrine Javorski-Miller and Ivan Delgado Orlic
A paper from 2008 mentions that quantitative PCR is 25 years old (VanGuilder et al., 2008) but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems (Gibson et al., 1996; Heid et al., 1996). Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instrument available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). In this chapter we highlight some key features that differentiate Real-Time PCR instruments, with the goal of simplifying the criteria needed to select the instrument that best fit a specific scientist's research needs.
7. qPCR Data Analysis: Unlocking the Secret to Successful Results
Jan Hellemans and Jo Vandesompele
Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. This chapter will cover all data analysis aspects from the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.
8. The MIQE Guidelines Uncloaked
Gregory L. Shipley
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.
9. PCR Applications for Epigenetics Research
Gavin Meredith, Miro Dudas, Mark Landers, Vasiliki Anest, Jonathan Wang, Caifu Chen, Peter Jozsi and Christopher Adams
The field of epigenetics transcends traditional genetics, genomics, molecular biology, and is poised to revolutionize the field of medical research and healthcare. It is a diverse field that encompasses the study of nuclear components such as chromatin structure, including histone modifications, protein/DNA interactions, protein/RNA interactions, and how these factors influence gene function. It also includes the study of DNA methylation and the role that non-coding RNAs play in influencing DNA methylation patterns, chromatin structure and ultimately regulating gene expression. Just as the field of epigenetics is broad and complex, so is the molecular technology of polymerase chain reaction (PCR). For every question one would like to address in any of these areas of epigenetics, there is a PCR application and instrumentation suitable to address it. For example there are numerous PCR-based approaches to look at DNA methylation patterns, densities, and even the methylation status of individual cytosine residues by PCR. Additionally, there are PCR methods to survey ncRNA expression and identify regions of the genome where proteins and RNA interact or where certain functional histone marks are located. This chapter provides an overview of these methodologies with a focus on the advantages and disadvantages of each approach.
10. High Resolution Melting Analysis
John F. Mackay and Carl T. Wittwer
Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA)- is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation. This chapter covers some of these developments and provides a guide to those wishing to establish this technique, as well as troubleshooting advice for those already underway.
11. Microfluidic Emulsion PCR
N. Reginald Beer and John H. Leamon
PCR has traditionally been performed in microliter-scale reactions because larger scale volumes are prohibitively expensive and wasteful while the smaller scales (nanoliter and below) are impractical with available sample handling tools and detection systems. At the microliter scale, samples can contain mutually competitive and distinct targets, introducing amplification bias and competitive inhibition that degrade assay performance. Microfluidic Emulsion PCR has emerged as a technique to resolve these challenges by a combination of two enabling technologies. Emulsion PCR provides the advantages of fluid partitioning, namely elimination of sample bias and the ability to run millions of reactions in discrete volumes, while microfluidics simultaneously reduces the sample volume, introduces a level of control over emulsion parameters, and provides optical observability of the partitioned microreactors. Furthermore, since microfluidic emulsions can be made monodisperse in size, they allow the assumption of an average dilution per reactor to permit the exploitation of Poisson statistics for very accurate titer estimation. Microfluidic emulsions can also be employed to perform solid-phase amplification with bead-based assays, combining yet another useful technique with the sample partitioning benefits of droplets. We expect the advantages of both emulsion PCR and microfluidics will encourage new applications and the integration of these enabling technologies will improve PCR performance.
How to buy this book
(EAN: 9781904455721 9781912530717 Subjects: [microbiology] [molecular microbiology] [bioinformatics] [genomics] [pcr] [molecular biology] [environmental microbiology] )