This newly revised second edition offers 71 protocols covering the many advances made in PCR technology since the first edition, which has been used in more than 10,000 laboratories worldwide. The book enables researchers at all levels to easily reproduce and adapt PCR experiments to their own specific requirements.
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 Kosel, Christoph B. Lucking, 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 Syvanen 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
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 It is what all molecular biology laboratories should have and contains information that is relevant to all levels of researchers. Relevant and timely diagrams compliment the text; there are also heaps of references included at the end of each section. Microbiologist