Polymerase Chain Reaction: Theory and Technology最新文献

{"title":"MIQE: Guidelines for the Design and Publication of a Reliable Real-time PCR Assay","authors":"J. Huggett, T. Nolan, S. Bustin","doi":"10.21775/9781912530243.12","DOIUrl":"https://doi.org/10.21775/9781912530243.12","url":null,"abstract":"The capacity to amplify and detect trace amounts of nucleic acids has made the polymerase chain reaction (PCR) the most formidable molecular technology in use today. Its versatility and scope was further broadened first with the development of reverse transcription (RT)-PCR, which opened up the entire RNA field to thorough exploration and then, most conspicuously, with its evolution into real-time quantitative PCR (qPCR). Speed, simplicity, specificity, wide linear dynamic range, multiplexing and high throughput potential, reduced contamination risk, simplified detection and data analysis procedures as well as availability of increasingly affordable instrumentation and reduced reagent cost have made qPCR the molecular method of choice when quantifying nucleic acids. Detection of pathogens, SNP analyses and quantification of RNA, even real-time analysis of gene expression in vivo have become routine applications and constant enhancements of chemistries, enzymes, mastermixes and instruments continue to extend the scope of qPCR technology by promising added benefits such as extremely short assay times measured in minutes, low reagent usage and exceptionally rapid heating/cooling rates. The whole process is driven by the insatiable demand for ever-more specific, sensitive, convenient and cost-effective protocols. However, it has also become clear that variable pre-assay conditions, poor assay design and incorrect data analysis have resulted in the regular publication of data that are often inconsistent, inaccurate and often simply wrong. The problem is exacerbated by a lack of transparency of reporting, with the details of technical information 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 design as well as recommendations to ensure comprehensive reporting of technical detail, indispensable conditions for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"3 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127776026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of mRNA Expression by Real-time PCR","authors":"S. Bustin, T. Nolan","doi":"10.21775/9781912530243.13","DOIUrl":"https://doi.org/10.21775/9781912530243.13","url":null,"abstract":"The last few years have witnessed the transformation of the real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) from an experimental technology into a mainstream scientific tool for the detection and quantification of RNA with an enormous range of uses in basic research, molecular medicine and biotechnology. The continuous improvement of reagents and instruments, combined with the trend towards high throughput and miniaturisation, is likely to reinforce that pre-eminence and continue to open up new application areas. Nonetheless, although in principle undoubtedly a straightforward technology, the reliability of RT-qPCR assays depends a series of sequential steps that include careful experimental design, optimisation and validation, which must be implemented pragmatically to obtain meaningful, biologically relevant data.","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"10 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115308852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oligonucleotide Primers and Probes: Use of Chemical Modifications to Increase or Decrease the Specificity of qPCR","authors":"S. Rose, R. Owczarzy, J. Dobosy, M. Behlke","doi":"10.21775/9781912530243.09","DOIUrl":"https://doi.org/10.21775/9781912530243.09","url":null,"abstract":"Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodi!ed DNA bases, selective use of chemically modi!ed bases and non-base modifying groups can prevent primer dimer artefacts, improve speci!city, and allow for selective ampli!cation of sequences that di\"er by as li#le as a single base. A wide variety of chemical modi!cations have been characterized for use in qPCR. As a general class, the modi!cations that are in greatest use today increase the binding a$nity of the oligonucleotides (i.e. increase the melting temperature, Tm). Tm-enhancing modi!cations allows both primers and probes to be shorter, improving the di\"erential Tm (ΔTm = Tm match – Tm mismatch) between perfect match and mismatch hybridization. &ese modi!cations have widespread application in allele-speci!c PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base modi!cations are in common use that decrease speci!city and improve duplex formation in the presence of base mismatches. Although these modi!cations lower Tm, they have less of an impact on primer stability than do actual mismatched bases. Universal bases permit use of primers and probes in polymorphic loci when it is desirable to detect all sequence variants and minimize mismatch discrimination. Introduction &e primers and probes used in real-time quantitative PCR (qPCR) are synthetic oligonucleotides and can be manufactured using natural DNA bases or can comprise RNA bases, arti!cially modi!ed bases, or a variety of pendant chemical groups not found in nature. While most needs are well met using unmodi!ed oligodeoxynucleotides, the use of chemical modi!cations can improve qPCR performance in a number of areas. &is chapter will cover three speci!c areas of interest, with a primary focus on increasing speci!city and allelic discrimination methods: 1 increasing speci!city, with a focus on SNP discrimination; 2 preventing primer-dimer or other PCR artefacts; 3 decreasing speci!city using universal bases. DOI: https://doi.org/10.21775/9781912530243.09","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"31 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123904484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Introduction to the Real-time Polymerase Chain Reaction","authors":"D. Rodríguez-Lázaro, M. Hernández","doi":"10.21775/9781912530243.01","DOIUrl":"https://doi.org/10.21775/9781912530243.01","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"233 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115091089","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantification Strategies in Real-time Polymerase Chain Reaction","authors":"M. Pfaffl","doi":"10.21775/9781912530243.05","DOIUrl":"https://doi.org/10.21775/9781912530243.05","url":null,"abstract":"The present chapter describes the quantification strategies used in real-time RT-PCR (RT-qPCR), focusing on the main elements that are essential to fulfil the MIQE guidelines. The necessity of initial proper data adjustment and background correction is discussed to allow reliable quantification. The advantages and disadvantages of the absolute and relative quantification approaches are also described. In conjunction with relative quantification, the importance of an amplification efficiency correction is shown, and software tools that are available to calculate relative expression changes are presented.","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"2014 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121323784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardization of Real-time PCR Methods in Food Microbiology","authors":"Kornelia Berghof-Jäger","doi":"10.21775/9781912530243.11","DOIUrl":"https://doi.org/10.21775/9781912530243.11","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126081196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Real-time PCR Arrays","authors":"N. Saunders","doi":"10.21775/9781912530243.14","DOIUrl":"https://doi.org/10.21775/9781912530243.14","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"129044496","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Homogenous Fluorescent Chemistries for Real-time PCR","authors":"M. A. Lee, D. Squirrell, D. Leslie, T. Brown","doi":"10.21775/9781912530243.03","DOIUrl":"https://doi.org/10.21775/9781912530243.03","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"95 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116880686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sample Preparation for Real-time PCR in Food Science","authors":"T. Kuchta","doi":"10.21775/9781912530243.07","DOIUrl":"https://doi.org/10.21775/9781912530243.07","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"82 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123091708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Internal and Other Controls for Real-time PCR Validation","authors":"M. A. Lee, D. Squirrell, D. Leslie","doi":"10.21775/9781912530243.08","DOIUrl":"https://doi.org/10.21775/9781912530243.08","url":null,"abstract":"","PeriodicalId":248876,"journal":{"name":"Polymerase Chain Reaction: Theory and Technology","volume":"10 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114901475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}