Phương pháp PCR 1. Khái niệm PCR (Polymerase Chain Reaction) (Phản ứng Trùng Phân, Phản ứng chuỗi Polymerase, hay phản ứng PCR)
PCRSecond Edition PCRSecond EditionMichael J. McPhersonInstitute of Molecular and Cellular Biology, Faculty of Biological Sciences,University of Leeds, Leeds, UKandSimon Geir MøllerFaculty of Science and TechnologyDepartment of Mathematics and Natural SciencesUniversity of StavangerN-4036 StavangerNorway Published by:Taylor & Francis GroupIn US: 270 Madison AvenueNew York, N Y 10016In UK: 4 Park Square, Milton ParkAbingdon, OX14 4RN© 2006 by Taylor & Francis GroupFirst published 2000; Second edition published 2006ISBN: 0-4153-5547-8 (Print edition)This book contains information obtained from authentic and highly regarded sources. Reprinted material isquoted with permission, and sources are indicated. A wide variety of references are listed. Reasonableefforts have been made to publish reliable data and information, but the author and the publisher cannotassume responsibility for the validity of all materials or for the consequences of their use.All rights reserved. No part of this book may be reprinted, reproduced, transmitted, or utilized in any formby any electronic, mechanical, or other means, now known or hereafter invented, including photocopying,microfilming, and recording, or in any information storage or retrieval system, without written permissionfrom the publishers.A catalog record for this book is available from the British Library.____________________________________________________________________Library of Congress Cataloging-in-Publication data has been applied for.Editor: Elizabeth OwenEditorial Assistant: Kirsty Lyons Production Editor: Karin HendersonTypeset by: Phoenix Photosetting, Chatham, Kent,UKPrinted by: MPG BOOKS Limited, Bodmin, Cornwall, UKTaylor & Francis Groupis the Academic Division of Informa UK LimitedVisit our web site at http://www.garlandscience.comThis edition published in the Taylor & Francis e-Library, 2006.“To purchase your own copy of this or any of Taylor & Francis or Routledge’scollection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”ISBN 0-203-00267-9 Master e-book ISBN ContentsAbbreviations ixPreface xiChapter 1 An Introduction to PCR 11.1 Introduction: PCR, a ‘DNA photocopier’ 11.2 PCR involves DNA synthesis 11.3 PCR is controlled by heating and cooling 31.4 PCR applications and gene cloning 51.5 History of PCR 6Chapter 2 Understanding PCR 92.1 How does PCR work? 92.2 PCR: a molecular perspective 112.3 The kinetics of PCR 152.4 Getting started 182.5 Post-PCR analysis 18Protocol 2.1: Basic PCR 20Chapter 3 Reagents and Instrumentation 233.1 Technical advances in PCR 233.2 Reagents 233.3 PCR buffers 233.4 Nucleotides 253.5 Modified nucleotides 253.6 PCR premixes 263.7 Oligonucleotide primers 263.8 DNA polymerases for PCR 363.9 Early PCR experiments 373.10 Thermostable DNA polymerases 373.11 Properties of Taq DNA polymerase 373.12 Thermostable proofreading DNA polymerases 433.13 Tth DNA polymerase has reverse transcriptase activity 463.14 Red and green polymerases and reagents 473.15 Polymerase mixtures: high-fidelity, long-range and RT-PCRs 483.16 Nucleic acid templates 513.17 Mineral oil 543.18 Plasticware and disposables 543.19 Automation of PCR and thermal cyclers 55Protocol 3.1: Phosphorylation of the 5′-end of an oligonucleotide 63 Chapter 4 Optimization of PCR 654.1 Introduction 654.2 Improving specificity of PCR 654.3 Template DNA preparation and inhibitors of PCR 754.4 Nested PCR improves PCR sensitivity 764.5 Contamination problems 764.6 Preventing contamination 804.7 Troubleshooting guide 82Chapter 5 Analysis, Sequencing and In Vitro Expression of PCR Products 875.1 Introduction 875.2 Analysis of PCR products 875.3 Verification of initial amplification product 895.4 Direct DNA sequencing of PCR products 935.5 Direct labeling of PCR products and homogenous assays 1015.6 In vitro expression of PCR product 103Protocol 5.1: Cycle sequencing – Applied Biosystems Big Dye terminators 108Chapter 6 Purification and Cloning of PCR Products 1116.1 Introduction 1116.2 Purification of PCR products 1116.3 Introduction to cloning of PCR products 1156.4 Approaches to cloning PCR products 1176.5 Confirmation of cloned PCR fragments 131Protocol 6.1: Blunt-end polishing of PCR fragments 134Protocol 6.2: PCR screening of bacterial colonies or cultures 135Chapter 7 PCR Mutagenesis 1377.1 Introduction 1377.2 Inverse PCR mutagenesis 1387.3 Unique sites elimination 1447.4 Splicing by overlap extension (SOEing) 1447.5 Point mutations 1507.6 Deletions and insertions 1517.7 Deletion mutagenesis 1517.8 Insertion mutagenesis 1517.9 Random mutagenesis 1577.10 PCR misincorporation procedures 1597.11 Recombination strategies 1607.12 RACHITT 1667.13 Gene synthesis 166Protocol 7.1: Inverse PCR mutagenesis 171Protocol 7.2: Quikchange mutagenesis of plasmid DNA 173Protocol 7.3: Splicing by overlap extension (SOEing) 175Protocol 7.4: ‘Sticky-feet’ mutagenesis 177vi Contents Protocol 7.5: DNA shuffling 179Protocol 7.6: Gene synthesis 182Chapter 8 Analysis of Gene Expression 1858.1 Introduction 1858.2 Reverse transcriptase PCR (RT-PCR) 1858.3 Semi-quantitative and quantitative RT-PCR 1898.4 One-tube RT-PCR 1948.5 Differential display 1948.6 PCR in a cell: in situ RT-PCR 1988.7 Microarrays 2048.8 RNA interference (RNAi) 205Protocol 8.1: Reverse transcriptase reaction 208Chapter 9 Real-Time RT-PCR 2099.1 Introduction 2099.2 Basic principles of real-time RT-PCR 2099.3 Detection methods 2129.4 General guidelines for probe and primer design 2219.5 Instruments and quantification of results 2229.6 Normalization and control selection 2259.7 A typical real-time RT-PCR experiment using SYBR® Green I 2259.8 Common real-time RT-PCR pitfalls 2289.9 Applications of real-time RT-PCR 229Chapter 10 Cloning Genes by PCR 233A Cloning genes of known DNA sequence 23310.1 Using PCR to clone expressed genes 23310.2 Express sequence tags (EST) as cloning tools 23710.3 Rapid amplification of cDNA ends (RACE) 238B Isolation of unknown DNA sequences 24010.4 Inverse polymerase chain reaction (IPCR) 24010.5 Multiplex restriction site PCR (mrPCR) 24310.6 Vectorette and splinkerette PCR 24410.7 Degenerate primers based on peptide sequence 248Protocol 10.1: 5′-RACE 253Protocol 10.2: Inverse PCR from plant genomic DNA 255Chapter 11 Genome Analysis 25711.1 Introduction 25711.2 Why map genomes? 25811.3 Single-strand conformation polymorphism analysis (SSCP) 25911.4 Denaturing-high-performance liquid chromatography (DHPLC) 26311.5 Ligase chain reaction (LCR) 26411.6 Amplification refractory mutation system (ARMS) 264Contents vii 11.7 Cleaved amplified polymorphic sequence analysis (CAPS) 26711.8 SNP genotyping using DOP-PCR 26811.9 Random amplified polymorphic DNA (RAPD) PCR 26911.10 Amplified fragment length polymorphisms (AFLPs) 27011.11 Multiplex PCR analysis of Alu polymorphisms 27011.12 Variable number tandem repeats in identity testing 27111.13 Minisatellite repeat analysis 27411.14 Microsatellites 27611.15 Sensitive PCR for environmental and diagnostic applications 27711.16 Screening transgenics 278viii Contents 8-MOP 8-methoxypsoralen8-oxo-dGTP 8-oxo-2′deoxyguanosineAFLP amplified lengthpolymorphismAMV avian myeloblastoma virusAP alkaline phosphataseAP-PCR arbitrarily primed PCRARMS amplification refractorymutation systemASA allele specific amplificationASP allele-specific PCRBAC bacterial artificialchromosomeBCIP 5-bromo, 4-chloro, 3-indolyl phosphateCAPS cleaved amplifiedpolymorphic sequenceanalysisCcdB control of cell deathCt threshold cycleCCD charge coupled device cDNA complementary DNACTcomparative threshold DHPLC denaturing-high-performance liquidchromatographyDIG digoxigeninDIG-dUTP digoxigenin-11-2′-deoxyuridine-5′-triphosphateDOP-PCR degenerate oligonucleotideprimed-PCRdPTP 6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][1,2]oxazin-7-oneELISA enzyme linkedimmunosorbent assayEST expressed sequence tagFAM 6-carboxyfluoresceinFDD fluorescent differentialdisplayFRET fluorescence resonanceenergy transferFS fluorescent sequencingGAPDH glyceraldehyde-3-phosphatedehydrogenaseGAWTS gene amplification withtranscript sequencingGM genetically modifiedHEX 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluoresceinHRP horseradish peroxidaseIPCR inverse polymerase chainreactionLCR ligase chain reactionLIC ligation-independentcloningM-MLV Moloney murine leukemiavirusMPSV mutations, polymorphismsand sequence variants mrPCR multiplex restriction sitePCRMVR minisatellite variant repeatNBT nitro blue tetrazoliumNF nonfluorescent nt nucleotidesORFs open reading framesPAGE polyacrylamide gelelectrophoresis PASA PCR amplification ofspecific alleles PBS phosphate buffered salinePCR polymerase chain reactionPCR-VNTRs PCR highly polymorphicvariable number tandemrepeatsPEETA Primer extension,Electrophoresis, Elution,Tailing, AmplificationPMBC peripheral bloodmononuclear cellsPMT photomultiplier tubeAbbreviations [...]... Introduction to PCR 1 1.1 Introduction: PCR, a ‘DNA photocopier’ 1 1.2 PCR involves DNA synthesis 1 1.3 PCR is controlled by heating and cooling 3 1.4 PCR applications and gene cloning 5 1.5 History of PCR 6 Chapter 2 Understanding PCR 9 2.1 How does PCR work? 9 2.2 PCR: a molecular perspective 11 2.3 The kinetics of PCR 15 2.4 Getting started 18 2.5 Post -PCR analysis 18 Protocol 2.1: Basic PCR 20 Chapter... (http://seq.yeastgenome.org/cgi-bin/web-primer); Primer3 26 PCR Protocol 7.5: DNA shuffling 179 Protocol 7.6: Gene synthesis 182 Chapter 8 Analysis of Gene Expression 185 8.1 Introduction 185 8.2 Reverse transcriptase PCR (RT -PCR) 185 8.3 Semi-quantitative and quantitative RT -PCR 189 8.4 One-tube RT -PCR 194 8.5 Differential display 194 8.6 PCR in a cell: in situ RT -PCR 198 8.7 Microarrays 204 8.8 RNA interference... Cloning Genes by PCR 233 A Cloning genes of known DNA sequence 233 10.1 Using PCR to clone expressed genes 233 10.2 Express sequence tags (EST) as cloning tools 237 10.3 Rapid amplification of cDNA ends (RACE) 238 B Isolation of unknown DNA sequences 240 10.4 Inverse polymerase chain reaction (IPCR) 240 10.5 Multiplex restriction site PCR (mrPCR) 243 10.6 Vectorette and splinkerette PCR 244 10.7 Degenerate... Post -PCR analysis 18 Protocol 2.1: Basic PCR 20 Chapter 3 Reagents and Instrumentation 23 3.1 Technical advances in PCR 23 3.2 Reagents 23 3.3 PCR buffers 23 3.4 Nucleotides 25 3.5 Modified nucleotides 25 3.6 PCR premixes 26 3.7 Oligonucleotide primers 26 3.8 DNA polymerases for PCR 36 3.9 Early PCR experiments 37 3.10 Thermostable DNA polymerases 37 3.11 Properties of Taq DNA polymerase 37 3.12 Thermostable... Real-Time RT -PCR 209 9.1 Introduction 209 9.2 Basic principles of real-time RT -PCR 209 9.3 Detection methods 212 9.4 General guidelines for probe and primer design 221 9.5 Instruments and quantification of results 222 9.6 Normalization and control selection 225 9.7 A typical real-time RT -PCR experiment using SYBR® Green I 225 9.8 Common real-time RT -PCR pitfalls 228 9.9 Applications of real-time RT -PCR 229 Chapter... understand and use PCR for experimental purposes. Although applications of PCR are provided these do not represent a comprehensive catalogue of all possible PCR applications, but serve to indicate the types of application possible. The main purpose of this new edition of PCR, as for the first edition, is to provide information on the fundamental principles of the reactions occurring in a PCR tube. Understanding... concept that Mullis had provided, and PCR became a major part of the business of Cetus, before they finally sold the rights to PCR in 1991 for $300m to Roche Molecular Systems. PCR and the thermostable polymerase responsible for the process were named as the first ‘Molecule of the Year’ in 1989 by the international journal Science. Since the myriad of applications of PCR were recognized it has become rather... testing. In particular PCR has become a central tool in the analysis and exploitation of genome sequence information, for example in gene knockout through RNA interference where PCR allows the rapid generation of appropriate constructs. It also facilitates measurement of levels of gene expression by ‘real-time’ PCR that monitors the level of product amplification at each cycle of the PCR (Chapter 9), providing... 10 11 product fragments after 20 cycles. In practice, as with most biological reactions, PCR amplification is not 100% efficient, so normally a greater number of cycles (25–40) are performed to achieve these levels of amplification. Understanding PCR 13 Table 2.1 Theoretical accumulation of PCR products during the first 20 cycles of a PCR with a single genomic DNA template Number of Number of Number of Cycle single... including promoter sequences to allow in vitro transcription of the PCR product, or sequences to allow the splicing or joining of PCR products (Chapter 7). A range of mutations can be introduced into a PCR product by altering the sequence of the primer (Chapter 7). The primers define the region of DNA to be amplified and can be used to tailor the PCR product for subsequent use. Reagents and instrumentation . PCR 6Chapter 2 Understanding PCR 92.1 How does PCR work? 92.2 PCR: a molecular perspective 112.3 The kinetics of PCR 152.4 Getting started 182.5 Post -PCR. transcriptase PCR (RT -PCR) 1858.3 Semi-quantitative and quantitative RT -PCR 1898.4 One-tube RT -PCR 1948.5 Differential display 1948.6 PCR in a cell: in situ RT-PCR