PCR Protocols

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.

1 A Short History of the Polymerase Chain Reaction.- 2 PCR Patent Issues.- 3 Equipping and Establishing a PCR Laboratory.- 4 Quality Control in PCR.- 5 Extraction of Nucleic Acid Templates.- 6 Extraction of DNA from Whole Blood.- 7 DNA Extraction from Tissue.- 8 Extraction of DNA from Microdissected Archival Tissues.- 9 RNA Extraction from Blood.- 10 RNA Extraction from Frozen Tissue.- 11 RNA Extraction from Tissue Sections.- 12 Dual DNA/RNA Extraction.- 13 DNA Extraction from Fungi, Yeast, and Bacteria.- 14 Isolation of RNA Viruses from Biological Materials.- 15 Extraction of Ancient DNA.- 16 DNA Extraction from Plasma and Serum.- 17 Technical Notes for the Detection of Nucleic Acids.- 18 Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels.- 19 PCR Primer Design.- 20 Optimization of Polymerase Chain Reactions.- 21 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.- 22 Rapid Amplification of cDNA Ends.- 23 Randomly Amplified Polymorphic DNA Fingerprinting: The Basics.- 24 Microsphere-Based Single Nucleotide Polymorphism Genotyping.- 25 Ligase Chain Reaction.- 26 Nested RT-PCR in a Single Closed Tube.- 27 Direct PCR from Serum: Application to Viral Genome Detection.- 28 Long PCR Amplification of Large Fragments of Viral Genomes: A Technical Overview.- 29 Long PCR Methodology.- 30 Qualitative and Quantitative PCR: A Technical Overview.- 31 Ultrasensitive PCR Detection of Tumor Cells in Myeloma.- 32 Ultrasensitive Quantitative PCR to Detect RNA Viruses.- 33 Quantitative PCR for cAMP RI Alpha mRNA: Use of Site-Directed Mutation and PCR Mimics.- 34 Quantitation of Multiple RNA Species.- 35 Differential Display: A Technical Overview.- 36 AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs.- 37 PCR Fluorescence Differential Display.- 38 Microarray Analysis Using RNA Arbitrarily Primed PCR.- 39 Oligonucleotide Arrays for Genotyping: Enzymatic Methods for Typing Single Nucleotide Polymorphisms and Short Tandem Repeats.- 40 Serial Analysis of Gene Expression.- 41 Mutation and Polymorphism Detection: A Technical Overview.- 42 Combining Multiplex and Touchdown PCR for Microsatellite Analysis.- 43 Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluorescence-Based Multiplex Microsatellite PCR.- 44 Reaction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA.- 45 Degenerate Oligonucleotide-Primed PCR.- 46 Mutation Detection Using RT-PCR-RFLP.- 47 Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms.- 48 PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA.- 49 Sequencing: A Technical Overview.- 50 Preparation and Direct Automated Cycle Sequencing of PCR Products.- 51 Nonradioactive PCR Sequencing Using Digoxigenin.- 52 Direct Sequencing by Thermal Asymmetric PCR.- 53 Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing.- 54 Direct Sequencing with Highly Degenerate and Inosine-Containing Primers.- 55 Determination of Unknown Genomic Sequences Without Cloning.- 56 Cloning PCR Products for Sequencing in M13 Vectors.- 57 DNA Rescue by the Vectorette Method.- 58 Technical Notes for Sequencing Difficult Templates.- 59 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.- 60 Cycling Primed In Situ Amplification.- 61 Direct and Indirect In Situ PCR.- 62 Reverse Transcriptase In Situ PCR: New Methods in Cellular Interrogation.- 63 Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes.- 64 Cloning and Mutagenesis: A Technical Overview.- 65 Using T4 DNA Polymerase to Generate Clonable PCR Products.- 66 A T-Linker Strategy for Modification and Directional Cloning of PCR Products.- 67 Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers.- 68 cDNA Libraries from a Low Amount of Cells.- 69 Creation of Chimeric Junctions, Deletions, and Insertions by PCR.- 70 Recombination and Site-Directed Mutagenesis Using Recombination PCR.- 71 Megaprimer PCR: Application in Mutagenesis and Gene Fusion.