Drawing on the proven qualities of the much praised and widely used first edition, John M. S. Bartlett and David Stirling have thoroughly updated and dramatically expanded the number of protocols to take advantage of the newest technologies used in all branches of research and clinical medicine today. These successful methods include real-time PCR, SNP analysis, nested PCR, direct PCR, and long-range PCR. Among the highlights are chapters on genome profiling by SAGE, differential display and chip technologies, the amplification of whole genome DNA by random degenerate oligonucleotide PCR, and the refinement of PCR methods for the analysis of fragmented DNA from fixed tissues. In situ PCR methods and their application in parallel with other methods, such as immunohistochemistry, are also included. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes a background introduction outlining the principle behind the technique, equipment and reagent lists, tips on troubleshooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results. Cutting-edge and highly practical, PCR Protocols, Second Edition provides both novice and experienced investigators with an up-to-date compendium of powerful PCR methods for easy reference and consultation in the day-to-day performance of PCR-based experimentation, one that will enhance understanding of PCR, satisfy current needs, and point to powerful future applications.
Reviews of the First Edition: "...succeeds in covering a wider range of more general applicable methods than...its predecessors ...provid[es] an extensive range of versatile, expedient, and readily applicable PCR protocols."-Trends in Cell Biology From reviews of the first edition... "...useful in virtually all disciplines of the life sciences...a pleasure [to] read and use..." -American Journal of Physiology: Lung Cellular and Molecular Physiology "...an excellent resource...has potential value for anyone looking for new ways to use PCR or new tricks to make their PCR work better." -Biopharm "...provides an extensive range of versatile, expedient, and readily applicable PCR protocols." -Trends in Cell Biology
Drawing on the highly successful first edition, this newly-revised edition covers the many advances made in PCR technology since the first book which has been used in more than 10,000 laboratories world wide.
Drawing on the highly successful first edition, this newly-revised second edition covers the many advances made in PCR technology since the first book, which has been used in more than 10,000 laboratories worldwide. As PCR technology has advanced significantly since the first edition, and has expanded its use in the clinical laboratory of physician/researchers, the scope of this book is greatly expanded to enable researchers at all levels to easily reproduce and adapt PCR experiments to their own specific requirements. The meethods selected represent worked examples from many fields that can be reproduced and adapted for use within the reader's laboratory. The authors have provided both a primer to allow the reader to gain basic experience of different PCR techniques, as well as in-depth insight into a variety of the more complex applications of PCR. This book will be essential for the labs of all biochemists, molecular biologists, geneticists and researchers utilizing the PCR techinque in their work.
Part I. Introduction to PCR
A Short History of the Polymerase Chain Reaction
John M. S. Bartlett and David Stirling
PCR Patent Issues
Peter Carroll and David Casimir
Equipping and Establishing a PCR Laboratory
Susan McDonagh
Quality Control in PCR
David Stirling
Part II. Preparation of Nucleic Acid Templates
Extraction of Nucleic Acid Templates
John M. S. Bartlett
Extraction of DNA from Whole Blood
John M. S. Bartlett and Anne White
DNA Extraction from Tissue
Helen Pearson and David Stirling
Extraction of DNA from Microdissected Archival Tissues
James J. Going
RNA Extraction from Blood
Helen Pearson
RNA Extraction from Frozen Tissue
John M. S. Bartlett
RNA Extraction from Tissue Sections
Helen Pearson
Dual DNA/RNA Extraction
David Stirling and John M. S. Bartlett
DNA Extraction from Fungi, Yeast, and Bacteria
David Stirling
Isolation of RNA Viruses from Biological Materials
Susan McDonagh
Extraction of Ancient DNA
Wera M. Schmerer
DNA Extraction from Plasma and Serum
David Stirling
Technical Notes for the Detection of Nucleic Acids
John M. S. Bartlett
Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels
David Stirling
Part III. Basic PCR Methods
PCR Primer Design
David L. Hyndman and Masato Mitsuhashi
Optimization of Polymerase Chain Reactions
Haiying Grunenwald
Subcycling PCR for Long-Distance Amplifications of Regions with High and Low Guanine-Cystine Content: Amplification of the Intron 22 Inversion of the FVIII Gene
David Stirling
Rapid Amplification of cDNA Ends
Xin Wang and W. Scott Young III
Randomly Amplified Polymorphic DNA Fingerprinting: The Basics
Ranil S. Dassanayake and Lakshman P. Samaranayake
Microsphere-Based Single Nucleotide Polymorphism Genotyping
Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li, Fei Ye, and Michael P. Weiner
Ligase Chain Reaction
William H. Benjamin, Jr., Kim R. Smith, and Ken B. Waites
Nested RT-PCR in a Single Closed Tube
Antonio Olmos, Olga Esteban, Edson Bertolini, and Mariano Cambra
Direct PCR from Serum: Application to Viral Genome Detection
Kenji Abe
Long PCR Amplification of Large Fragments of Viral Genomes: A Technical Overview
Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell
Long PCR Methodology
Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell
Part IV. Ultrasensitive and Quantitative PCR
Qualitative and Quantitative PCR: A Technical Overview
David Stirling
Ultrasensitive PCR Detection of Tumor Cells in Myeloma
Friedrich W. Cremer and Marion Moos
Ultrasensitive Quantitative PCR to Detect RNA Viruses
Susan McDonagh
Quantitative PCR for cAMP RI Alpha mRNA: Use of Site-Directed Mutation and PCR Mimics
John M. S. Bartlett
Quantitation of Multiple RNA Species
Ron Kerr
Part V. Transcriptome Analysis
Differential Display: A Technical Overview
John M. S. Bartlett
AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs
Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab, Eva Belloso, and Ricardo Pujol-Borrell
PCR Fluorescence Differential Display
Kostya Khalturin, Sergej Kuznetsov, and Thomas C. G. Bosch
Microarray Analysis Using RNA Arbitrarily Primed PCR
Steven Ringquist, Gaelle Rondeau, Rosa-Ana Risques, Takuya Higashiyama, Yi-Peng Wang, Steffen Porwollik, David Boyle, Michael McClelland, and John Welsh
Oligonucleotide Arrays for Genotyping: Enzymatic Methods for Typing Single Nucleotide Polymorphisms and Short Tandem Repeats
Stephen Case-Green, Clare Pritchard, and Edwin Southern
Serial Analysis of Gene Expression, Karin A. Oien
Part VI. Mutations and Polymorphisms
Mutation and Polymorphism Detection: A Technical Overview
Joanne Edwards and John M. S. Bartlett
Combining Multiplex and Touchdown PCR for Microsatellite Analysis
Kanokporn Rithidech and John J. Dunn
Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluorescence-Based Multiplex Microsatellite PCR
Joanne Edwards and John M. S. Bartlett
Reduction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA
Wera M. Schmerer
Degenerate Oligonucleotide-Primed PCR
Michaela Aubele and Jan Smida
Mutation Detection Using RT-PCR-RFLP
Hitoshi Nakashima, Mitsuteru Akahoshi, and Yosuke Tanaka
Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms
David Stirling
PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA
Mei Han and Mary Ann Robinson
Part VII. PCR-Based Sequencing
Sequencing: A Technical Overview
David Stirling
Preparation and Direct Automated Cycle Sequencing of PCR Products
Susan E. Daniels
Nonradioactive PCR Sequencing Using Digoxigenin
Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger, and Manuel B. Graeber
Direct Sequencing by Thermal Asymmetric PCR
Georges-Raoul Mazars and Charles Theillet
Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing
Anu Suomalainen and Ann-Christine Syvänen
Direct Sequencing with Highly Degenerate and Inosine-Containing Primers
Zhiyuan Shen, Jingmei Liu, Robert L. Wells, and Mortimer M. Elkind
Determination of Unknown Genomic Sequences Without Cloning
Jean-Pierre Quivy and Peter B. Becker
Cloning PCR Products for Sequencing in M13 Vectors
David Walsh
DNA Rescue by the Vectorette Method
Marcia A. McAleer, Alison J. Coffey, and Ian Dunham
Technical Notes for Sequencing Difficult Templates
David Stirling
Part VIII. In Situ PCR and PRINS
PCR-Based Detection of Nucleic Acids in Chromosomes, Cells, and Tissues: Technical Considerations on PRINS and In Situ PCR, and Comparison with In Situ Hybridization
Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman
Cycling Primed In Situ Amplification
John H. Bull and Lynn Paskins
Direct and Indirect In Situ PCR
Klaus Hermann Wiedorn and Torsten Goldmann
Reverse Transcriptase In Situ PCR: New Methods in Cellular Interrogation
Mark Gilchrist and A. Dean Befus
Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes
Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman
Part IX. Cloning and Mutagenesis
Cloning and Mutagenesis: A Technical Overview
Helen Pearson and David Stirling
Using T4 DNA Polymerase to Generate Clonable PCR Products
Kai Wang
A T-Linker Strategy for Modification and Directional Cloning of PCR Products
Robert M. Horton, Raghavanpillai Raju, and Bianca M. Conti-Fine
Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers
Gregory M. Preston
cDNA Libraries from a Low Amount of Cells
Philippe Ravassard, Christine Icard-Liepkalns, Jacques Mallet, and Jean Baptiste Dumas Milne Edwards
Creation of Chimeric Junctions, Deletions, and Insertions by PCR
Genevieve Pont-Kingdon
Recombination and Site-Directed Mutagenesis Using Recombination PCR
Douglas H. Jones and Stanley C. Winistorfer
Megaprimer PCR: Application in Mutagenesis and Gene Fusion
Emily Burke and Sailen Barik
Index