Polymerase Chain Reaction How to Read Results

Polymerase Chain Reaction: Theory and Technology

Publisher: Caister Bookish Press
Writer: Marking A. Behlke, Kornelia Berghof-Jäger, Tom Brown, et al.

Pages: vi +262
Paperback:
Publication engagement: July 2019
ISBN: 978-1-912530-24-three
Price: GB £159 or Usa $319Buy book or Purchase online
Ebook:
Publication date: July 2019
ISBN: 978-one-912530-25-0
Price: U.s. $319Buy ebook
DOI: https://doi.org/10.21775/9781912530243

The polymerase chain reaction (PCR) is a powerful research tool used in many scientific disciplines. It is also used for detection and testing in areas such as food microbiology, ecology microbiology, biotechnology, industrial microbiology, veterinary and medical diagnostics.

This indispensable manual is a compilation of review articles written by experts in the field of PCR applied science. Topics covered include: principles of PCR, fluorescent chemistries, instrumentation, quantification strategies, extraction and purification of nucleic acids, sample preparation, controls for validation, primers and probes, standardization of methods, MIQE guidelines, mRNA expression and PCR arrays.

This volume provides a comprehensive overview of PCR theory, instrumentation and methods. The book represents an excellent, detailed guide for anyone interested in the development and apply of PCR technology. Information technology is a recommended purchase for all microbiology and molecular biology laboratories and academy libraries.

Table of contents

1. Introduction to the Real-time Polymerase Chain Reaction

David Rodríguez-Lázaro and Marta Hernández

Pages: i-xviii.

Food safety and quality control programs are increasingly applied throughout the product food chain in order to guarantee added value products as well as to minimize the gamble of infection for the consumer. The development of real-time PCR has represented ane of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly of import for the agronomical and nutrient industry. Dissimilar strategies for real-time PCR diagnostic take been developed including unspecific detection contained of the target sequence using fluorescent dyes such as SYBR Greenish, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

2. Principles of the Real-time Polymerase Chain Reaction

Stephen A Bustin, Sara Zaccara and Tania Nolan

Pages: 19-42.

The real-fourth dimension fluorescence-based quantitative polymerase concatenation reaction (qPCR) has become the benchmark engineering for the detection of nucleic acids in every surface area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative stop-bespeak assay, qPCR allows accurate quantification of amplified Dna in real time during the exponential stage of the reaction. The toll of instruments and reagents is well within reach of individual laboratories, assays are piece of cake to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to attain accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR analysis, starting with sample pick, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the meaning bug associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE).

iii. Homogenous Fluorescent Chemistries for Real-time PCR

Martin A. Lee, David J. Squirrell, Dario Fifty. Leslie and Tom Brown

Pages: 43-78.

The development of fluorescent methods for the airtight tube polymerase concatenation reaction has greatly simplified the process of quantification. Current approaches employ fluorescent probes that interact with the distension products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to Deoxyribonucleic acid quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that utilize the phenomenon of Fluorescent Energy Transfer. In this chapter nosotros describe these methods in detail, outline the principles of each procedure, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to testify how they may exist used on various commercially available thermal cyclers.

4. Instrumentation and Fluorescent Chemistries Used in Quantitative Polymerase Chain Reaction

Mathilde H. Josefsen, Charlotta Löfström, Trine Hansen, Eyjólfur Reynisson and Jeffrey Hoorfar

Pages: 79-104.

The polymerase chain reaction has revolutionized the earth of scientific research and its broad awarding has acquired a tremendous evolution of versatile PCR instruments and chemistries to fit its purpose. This chapter provides the reader with a general introduction to the basics of real-time PCR instrumentation, including the thermal and optical systems and the software. Performance parameters such as temperature uniformity, accurateness and ramp speed as well equally reaction format, optical systems, scale of dyes, software and comparison between unlike real-fourth dimension PCR platforms will be discussed from a user perspective leading to an instrument choice guide. Differences between fluorescent Deoxyribonucleic acid bounden dyes and target-specific fluorescently labeled primers or probes for detection of amplicon aggregating volition be discussed, forth with the properties and applications of the most frequently applied chemistries. The fluorophores and quenchers used for primer and probe labeling and their compatibility volition be presented, and finally the futurity challenges and trends within the field of qPCR instrumentation will be discussed.

five. Quantification Strategies in Real-fourth dimension Polymerase Chain Reaction

Michael W. Pfaffl

Pages: 105-114.

The nowadays chapter describes the quantification strategies used in real-fourth dimension RT-PCR (RT-qPCR), focusing on the chief elements that are essential to fulfil the MIQE guidelines. The necessity of initial proper information adjustment and background correction is discussed to permit reliable quantification. The advantages and disadvantages of the absolute and relative quantification approaches are as well described. In conjunction with relative quantification, the importance of an amplification efficiency correction is shown, and software tools that are bachelor to calculate relative expression changes are presented.

half dozen. The Extraction and Purification of Nucleic Acids for Assay by PCR

Chaminda Salgado and Waqar Hussain

Pages: 115-126.

Myriad methods for the extraction and purification of nucleic acids prior to PCR are currently used throughout the community. While these methods accept many unique and bespoke aspects, they broadly follow a sequence of lysis, isolation, washing and elution to get from a complex biological sample to purified nucleic acid that can be used in a PCR reaction. Various common methods available for each stage are described and potential sequences for particular sample types can be discerned. The potential for these methods to be automated are discussed and the process options summarized with respect to the speed of the methods, technical skill required and the resultant purity and yield that can be expected.

7. Sample Grooming for Existent-time PCR in Nutrient Science

Tomáš Kuchta

Pages: 127-136.

Sample grooming including Deoxyribonucleic acid isolation is described as the first procedural step preceding the analysis of food by real-time PCR. Principles of, applications of and prerequisites for direct DNA isolation from food matrix are presented, providing information on chaotropic solid stage extraction, solubilization with cetyltrimethylammonium bromide followed by liquid-liquid extraction, and on immunomagnetic separation. Importance of and procedures for testing the amplifiability of the isolated DNA, conclusion of the recovery and recovery charge per unit of Dna from food too as procedures improving the efficiency of DNA isolation from "difficult" food matrices are given. Various techniques for DNA quantitation, including UV spectrometry, fluorimetry and determination of amplifiable Dna past PCR, are described and their utilise and usefulness are discussed. The effectivity of private approaches at the analysis of various nutrient matrices is discussed. For the application field of rapid detection of pathogenic bacteria in food, data on cultivation enrichment, immunoseparation, centrifugation and microfiltration is provided. The applicability of dissimilar sample preparation methods at the detection of various pathogenic bacteria in several food product types is discussed. Attention is paid too to DNA isolation from enriched samples, including clarification of rapid techniques for partial Dna separation, bacterial cell lysis and removal of PCR inhibitors.

eight. Internal and Other Controls for Existent-time PCR Validation

Martin A. Lee, David J. Squirrell and Dario Fifty. Leslie

Pages: 137-150.

A range of factors can cause false negative results in real-time PCR through effects on one or more of the reaction components. Consequently applications requiring a high level of confidence need to be designed to control for the occurrence of false negatives. Whilst an external, or batch, control is frequently used, the platonic control is an internal one included in the reaction cocktail in a multiplex assay. Hither we talk over the application and development of molecular mimics as controls in real-fourth dimension PCR and explicate concepts and experimental considerations to help in the optimisation of controlled multiplexed assays.

9. Oligonucleotide Primers and Probes: Use of Chemic Modifications to Increase or Decrease the Specificity of qPCR   Costless download

Scott D. Rose, Richard Owczarzy, Joseph R. Dobosy and Mark A. Behlke

Pages: 151-178.

Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodified Dna bases, selective use of chemically-modified bases and non-base modifying groups can prevent primer-dimer artifacts, improve specificity, and allow for selective amplification of sequences that differ by every bit little as a unmarried base. A wide diversity of chemical modifications take been characterized for apply in qPCR. Every bit a general course, the modifications that are in greatest use today increase the binding affinity of the oligonucleotides (i.e., increase the melting temperature, Tm). Tm-enhancing modifications allows both primers and probes to be shorter, improving the differential Tm (ΔTm=Tm lucifer-Tm mismatch) between perfect friction match and mismatch hybridization. These modifications have widespread application in allele-specific PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base of operations modifications are in common use that decrease specificity and meliorate duplex formation in the presence of base of operations mismatches. Although these modifications lower Tm, they have less of an impact on primer stability than exercise actual mismatched bases. Universal bases allow employ of primers and probes in polymorphic loci when it is desirable to find all sequence variants and minimize mismatch discrimination.

x. Internal Distension Controls in Real-time Polymerase Chain Reaction-Based Methods for Pathogen Detection

Nigel Cook, Gabriel A de Ridder, Martin D'Agostino and Maureen B Taylor

Pages: 179-186.

Assays based on nucleic acrid amplification are highly efficient, but they tin can be afflicted past the presence of matrix-derived substances which can interfere or prevent the reaction from performing correctly. Careful sample treatment must exist practical/used to remove these inhibitory substances. Still no sample treatment can exist relied on completely, thus an amplification command should exist employed to be able to verify that the assay has performed correctly. An internal amplification command (IAC) is a not-target DNA sequence present in the very same reaction equally the sample or target nucleic acid extract. If it is successfully amplified to produce a signal, whatever non-production of a target signal in the reaction is considered to signify that the sample did not contain the target pathogen or organism. If notwithstanding the reaction produces neither a betoken from the target nor the IAC, it signifies that the reaction has failed.

11. Standardization of Real-time PCR Methods in Food Microbiology

Kornelia Berghof-Jäger

Pages: 187-198.

International law requires that only food suitable for consumption may achieve the market. To meet this demand, thorough microbiological testing must be performed on raw materials, the manufacturing process and finished products. Real-time PCR methods are particularly well-suited for this testing as they are fast, precise and very specific. Multiple methods, including real-time PCR, exist for testing the same analyte. These are favored according to regional preferences and regulatory requirements. Withal, global trade could be simplified if there was an international consensus on a set of belittling standards. The International Organization for Standardization (ISO) and the European Organization for Standardization (CEN) are platforms for generating standards through open, counterbalanced and consensus-driven processes. To avoid duplication of work and structures, an agreement on technical co-operation between ISO and CEN (Vienna Understanding) was canonical in 1991. This allows for focused expertise to be used efficiently to benefit international standardization. Currently, a few full general Standards exist which describe the basic requirements of PCR methods. Full general standardization documents focusing on performance characteristics as well as basic requirements and definitions of existent-time PCR are in development. Standards for specific detection of the nutrient-borne pathogens Clostridium botulinum, Yersinia, STEC, Vibrio and viruses are also in progress. In parallel, standardization of real-time PCR-based methods for the detection of genetically modified organisms (GMO) and allergens in food are ongoing.

12. MIQE: Guidelines for the Design and Publication of a Reliable Real-time PCR Assay

Jim Huggett, Tania Nolan and Stephen A. Bustin

Pages: 199-210.

The capacity to amplify and discover trace amounts of nucleic acids has fabricated the polymerase chain reaction (PCR) the most formidable molecular technology in use today. Its versatility and scope was further broadened showtime with the development of reverse transcription (RT)-PCR, which opened upwardly the entire RNA field to thorough exploration and so, about clearly, with its development into existent-time quantitative PCR (qPCR). Speed, simplicity, specificity, wide linear dynamic range, multiplexing and high throughput potential, reduced contamination take chances, simplified detection and data analysis procedures as well as availability of increasingly affordable instrumentation and reduced reagent price have made qPCR the molecular method of choice when quantifying nucleic acids. Detection of pathogens, SNP analyses and quantification of RNA, even existent-time analysis of gene expression in vivo take go routine applications and constant enhancements of chemistries, enzymes, mastermixes and instruments continue to extend the scope of qPCR engineering by promising added benefits such as extremely short assay times measured in minutes, low reagent usage and exceptionally rapid heating/cooling rates. The whole procedure is driven past the insatiable need for ever-more specific, sensitive, convenient and toll-effective protocols. Notwithstanding, it has also get clear that variable pre-assay conditions, poor assay pattern and incorrect data analysis have resulted in the regular publication of information that are frequently inconsistent, inaccurate and often simply wrong. The problem is exacerbated by a lack of transparency of reporting, with the details of technical data wholly inadequate for the purpose of assessing the validity of reported qPCR data. This has serious consequences for basic research, reducing the potential for translating findings into valuable applications and potentially devastating implications for clinical practice. In response, guidelines proposing a minimum standard for the provision of information for qPCR experiments (MIQE) have been launched. These aim to establish a standard for accurate and reliable qPCR experimental blueprint as well as recommendations to ensure comprehensive reporting of technical detail, indispensable conditions for the maturing of qPCR into a robust, authentic and reliable nucleic acid quantification technology.

thirteen. Analysis of mRNA Expression by Real-time PCR

Stephen A. Bustin and Tania Nolan

Pages: 211-248.

The last few years take witnessed the transformation of the real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) from an experimental applied science into a mainstream scientific tool for the detection and quantification of RNA with an enormous range of uses in basic inquiry, molecular medicine and biotechnology. The continuous comeback of reagents and instruments, combined with the trend towards loftier throughput and miniaturisation, is likely to reinforce that pre-eminence and continue to open up new awarding areas. Withal, although in principle undoubtedly a straightforward engineering, the reliability of RT-qPCR assays depends a serial of sequential steps that include careful experimental design, optimisation and validation, which must be implemented pragmatically to obtain meaningful, biologically relevant data.

14. Real-time PCR Arrays

Nick A. Saunders

Pages: 249-262.

Real-fourth dimension PCR arrays are tools that permit convenient testing of samples in many assays concurrently, parallel testing of many samples or testing of multiple samples and targets simultaneously. Information technology is desirable to standardise and automate primer and probe pick due to the big number of assays that must be designed. Furthermore, it is useful to use probe selection techniques that increase the robustness of the individual assays since this will increase the level of compatibility between the assays and decrease the complexity of interpretation of the outputs. A elementary approach to creating existent-time PCR arrays is to use microtitre plates which currently have capacities of 96, 384 or 1536 features. Such arrays can be populated with user designed assays or with tests selected grade a carte of over one million that are commercially available. A primary application of such arrays has been to verify gene expression data obtained using hybridisation. Cramming boosted features into a device of manageable scale has led to the introduction of nanolitre volume arrays that diverge from the microtitre plate pattern. Several thousand dissimilar reactions can now be included in a single real-time PCR array. The reduction in scale also has advantages in terms of the volumes of materials required. As existent-time arrays are miniaturised the number of pipetting steps required increases and information technology is often necessary to pre-configure them commercially leading to relative inflexibility. This limitation has prompted the evolution of arrays that include microfluidic channels and valves. These 'chips' can exist loaded via relatively few liquid treatment steps to create custom applications.

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(EAN: 9781912530243 9781912530250 Subjects: [molecular biology] [molecular microbiology] [pcr] )

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