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E-raamat: PCR: Methods Express

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Edited by Simon Hughes, Edited by Adrian Moody

Formaat: 348 pages
Sari: Life Science
Ilmumisaeg: 01-May-2007
Kirjastus: Scion Publishing Ltd
Keel: eng
ISBN-13: 9781907904424

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Formaat: 348 pages
Sari: Life Science
Ilmumisaeg: 01-May-2007
Kirjastus: Scion Publishing Ltd
Keel: eng
ISBN-13: 9781907904424

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PCR: Methods Express describes the very latest PCR-based methodologies and approaches to provide the most up-to-date practical advice on how to tackle a broad range of biological problems.

PCR is the most widely used technique in molecular biology. New PCR variants offering substantial benefits to existing protocols appear on a frequent basis. PCR: Methods Express describes the very latest PCR-based methodologies and approaches to provide the most up-to-date practical advice on how to tackle a broad range of biological problems including:

*real time qRT-PCR
*rapid generation of gene targeting constructs
*PCR multiplexes
*PCR-based mutagenesis
*identification of microdeletions and microduplications

*DNA methylation analysis
*forensic genetic DNA typing
*genotyping
*identification of mutations in single cells
*whole genome amplification
*diagnosis of infectious diseases
*inverse PCR-based RFLP

This book is a comprehensive research guide; every chapter discusses the merits and limitations of the available approaches and then provides fully-proven protocols with hints and tips for success. PCR: Methods Express is an essential laboratory manual for researchers in all life science fields and at all levels, from postgraduate student to principal investigator.

Contributors

xiii

Foreword

xvii

Preface

xviii

Abbreviations

xix

Color section

xxi

An introduction to the polymerase chain reaction

Adrian Moody

Introduction

(3)

History of PCR

(2)

Utility of PCR - modifications and applications

(1)

Methods and approaches

(14)

Components of PCR

(1)

The PCR cycle

(1)

Thermal cyclers

(1)

Oligonucleotide primers

(2)

Standard PCR

(3)

Optimizing a PCR

(2)

Optimizing MgCl2 concentration

(4)

General hints and tips

(1)

Troubleshooting

(2)

References

(1)

Polymerases for PCR

Meg Martel

Simon Baker

Ian Kavanagh

Simon May

Introduction

(1)

Function of DNA polymerases

(1)

Origins of DNA polymerases

(1)

Methods and approaches

(11)

Unit definition of DNA polymerase

(1)

DNA polymerase activity

(1)

Choice of enzyme

(2)

Hot-start PCR

(2)

Buffer composition

(1)

Inhibitors

(1)

Detection methods for assessing enzyme activity

(6)

Troubleshooting

(1)

References

(2)

A detailed guide to quantitative RT-PCR

Pete Kaiser

Introduction

(1)

Methods and approaches

(10)

Technologies available

(2)

Choice of reference gene

(1)

Analysis of real-time qRT-PCR data

(3)

Designing TaqMan primers and probes

(1)

Recommended protocols

(2)

Analyzing primer optimization results

(3)

Troubleshooting

(1)

References

(2)

Use of quantitative PCR for the detection of genomic microdeletions or microduplications

Simon Hughes

Rosanna Weksberg

Laura Moldovan

Jeremy A. Squire

Introduction

(2)

Test model

(1)

Methods and approaches

(10)

SYBR Green

(1)

Measuring amplification

(2)

Primer design

(1)

Housekeeping/reference genes and data normalization

(1)

Primer optimization

(1)

Generating a standard curve

(2)

Recommended protocols

(2)

DNA quantification and data analysis

(1)

Case study

(2)

Concluding remarks

(1)

Troubleshooting

(1)

References

(1)

Robust and unique PCR for single-nucleotide polymorphism genotyping applications

Xiangning Chen

Introduction

(2)

Variable number of tandem repeats

(1)

Single-nucleotide polymorphisms

(1)

Methods and approaches

(12)

General guidelines for genotyping applications

(3)

SNP genotyping methods

(2)

General considerations

(1)

Recommended protocols

(5)

Typical results

(1)

Summary

(1)

Troubleshooting

(2)

References

(2)

Using PCR and linkage mapping to identify single genes and quantitative trait loci for livestock traits

Jillian F. Maddox

Imke Tammen

Sonja Dominik

Introduction

(2)

Genetic traits

(1)

Genetic markers

(1)

Methods and approaches

(20)

Genotyping

(17)

Data analysis: linkage analysis and trait mapping

100

(2)

Definition of terms

102

(1)

Troubleshooting

103

(1)

References

104

(3)

PCR restriction fragment length polymorphism analysis for genotyping of single-nucleotide polymorphisms

Simon Hughes

Introduction

107

(2)

Restriction enzymes

107

(1)

RFLP

108

(1)

PCR-RFLP

108

(1)

Methods and approaches

109

(10)

Restriction enzyme identification

110

(6)

DNA extraction and PCR amplification

116

(3)

Results and data interpretation

119

(1)

Troubleshooting

119

(2)

References

121

(2)

Forensic genetic DNA typing with PCR-based methods

Clous Børsting

Juan J. Sanchez

Niels Morling

Introduction

123

(2)

PCR and forensic genetics

123

(1)

Mitochondrial DNA analysis

124

(1)

Single nucleotide polymorphisms

125

(1)

Methods and approaches

125

(15)

PCR fragment analysis

125

(1)

PCR optimization

125

(1)

PCR multiplexing

126

(1)

Recommended protocols

127

(13)

Troubleshooting

140

(1)

References

141

(2)

Large PCR multiplexes with special reference to forensic single-nucleotide polymorphism typing

Juan J. Sanchez

Claus Børsting

Niels Morling

Introduction

143

(3)

Genetic markers

143

(1)

SNP typing

144

(1)

SNP multiplexing

145

(1)

Methods and approaches

146

(10)

Multiplex design

146

(2)

Recommended protocols

148

(5)

Multiple-injection protocol

153

(3)

Troubleshooting

156

(1)

References

157

(2)

Pre-implantation genetic diagnosis of monogenic disease: PCR-based methods for the identification of mutations in single cells

Dagan Wells

Introduction

159

(6)

Principles of PGD

159

(1)

Contamination

160

(2)

Amplification efficiency

162

(1)

Allele dropout

163

(1)

Whole genome amplification

163

(2)

Methods and approaches

165

(5)

Expected results from multiplex PCR

169

(1)

Troubleshooting

170

(1)

References

171

(2)

Rapid generation of gene-targeting constructs

Trevor J. Wilson

Dirk Truman

Antonietta Giudice

Paul Hertzog

Introduction

173

(3)

The function (or phenotype)-driven approach

173

(1)

The gene-driven approach

174

(2)

Methods and approaches

176

(19)

Obtaining gene sequences and identification of BACs

178

(4)

ET cloning procedure

182

(3)

Cloning of floxed exon

185

(1)

Preparation and electroporation of DNA

186

(9)

Screening of ES cell clones

195

(1)

Troubleshooting

195

(1)

References

196

(1)

Construction of long DNA molecules from multiple fragments using PCR

Nikolai A. Shevchuk

Anton V. Bryksin

Introduction

197

(1)

Methods and approaches

197

(17)

Principles of long multiple fusion

197

(2)

Limitations of long multiple fusion

199

(1)

Factors critical for successful long multiple fusion

200

(3)

Recommended protocols

203

(11)

Troubleshooting

214

(1)

References

214

(3)

Efficient PCR-based mutagenesis method applicable to diverse mutagenesis strategies using type lls restriction enzymes

Jae-Kyun Ko

Jianjie Ma

Introduction

217

(1)

Methods and approaches

218

(7)

Principles of mutagenesis

218

(1)

Design of mutagenic primers and choice of type lls restriction enzyme for mutagenesis

219

(5)

Application to diverse mutagenesis

224

(1)

Summary

225

(1)

Troubleshooting

225

(3)

References

228

(1)

Inverse PCR-based restriction fragment length polymorphism for identifying low-level mutations in tumors

G. Mike Makrigiorgos

Introduction

229

(1)

Methods and approaches

230

(10)

Principles of inverse PCR-based amplified restriction fragment length polymorphism

230

(6)

Size-separation analysis of PCR products and estimation of mutation frequencies

236

(1)

Sample results

236

(4)

Troubleshooting

240

(1)

References

240

(3)

PCR methods for infectious disease diagnosis

Padmini Ramachandran

Andrew Hardick

Charlotte Gaydos

Samuel Yang

Richard Rothman

Introduction

243

(2)

PCR for infectious disease diagnosis in a clinical setting

244

(1)

Antimicrobial resistance profiling

245

(1)

PCR and biodefense

245

(1)

Methods and approaches

245

(16)

Cost of PCR

245

(1)

False-negative and -positive results

246

(1)

Sample processing for PCR

247

(1)

Sample preparation from various human specimens

247

(2)

Recommended protocols

249

(12)

Troubleshooting

261

(2)

References

263

(2)

Use of PCR for DNA methylation analyses

Mario F. Fraga

Manel Esteller

Introduction

265

(1)

Methods and approaches

266

(9)

PCR-based techniques

266

(9)

Summary

275

(1)

Troubleshooting

275

(2)

References

277

(2)

PCR-based methods to determine DNA methylation status at specific CpG sites using methylation-sensitive restriction enzymes

Helmtrud I. Roach

Ko Hashimoto

Introduction

279

(1)

Methods and approaches

280

(10)

Methylation-sensitive restriction enzymes

280

(1)

Principle of the MSRE PCR method

281

(1)

Identifying CpG sites and suitable MSREs

282

(1)

Extraction of nucleic acids

283

(3)

Detection of methylation status using MSREs

286

(3)

Applications

289

(1)

Troubleshooting

290

(1)

References

291

(2)

PCR-based whole genome amplification

Nona Arneson

Simon Hughes

Richard Houlston

Susan Done

Introduction

293

(1)

Methods and approaches

294

(20)

DOP-PCR

296

(3)

PEP-PCR

299

(2)

Ligation-mediated PCR

301

(12)

Downstream applications

313

(1)

Troubleshooting

314

(2)

General troubleshooting

314

(2)

I-PEP-PCR

316

(1)

PRSG

316

(1)

References

316

(3)

PCR sequencing of human genes for the discovery of DNA sequence variants

Abizar Lakdawalla

Introduction

319

(1)

DNA sequence variations

319

(1)

Methods and approaches

320

(20)

Resequencing methods

320

(14)

Analysis of results

334

(5)

Mutation nomenclature

339

(1)

Troubleshooting

340

(2)

References

342

(1)

Appendix 1 List of suppliers

343

(2)

Index

345

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