Pcr Protocols

by Bartlett, John; Stirling, David

• ISBN13: 9780896036277
• ISBN10: 0896036278
• Edition: 2nd
• Format: Paperback
• Copyright: 2003-08-01
• Publisher: Humana Pr Inc

Summary

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.

Table of Contents

Preface	p. v
Contributors	p. xiii
Introduction to PCR
A Short History of the Polymerase Chain Reaction	p. 3
PCR Patent Issues	p. 7
Equipping and Establishing a PCR Laboratory	p. 15
Quality Control in PCR	p. 21
Preparation of Nucleic Acid Templates
Extraction of Nucleic Acid Templates	p. 27
Extraction of DNA from Whole Blood	p. 29
DNA Extraction from Tissue	p. 33
Extraction of DNA from Microdissected Archival Tissues	p. 35
RNA Extraction from Blood	p. 43
RNA Extraction from Frozen Tissue	p. 45
RNA Extraction from Tissue Sections	p. 47
Dual DNA/RNA Extraction	p. 49
DNA Extraction from Fungi, Yeast, and Bacteria	p. 53
Isolation of RNA Viruses from Biological Materials	p. 55
Extraction of Ancient DNA	p. 57
DNA Extraction from Plasma and Serum	p. 63
Technical Notes for the Detection of Nucleic Acids	p. 65
Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels	p. 77
Basic PCR Methods
PCR Primer Design	p. 81
Optimization of Polymerase Chain Reactions	p. 89
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	p. 101
Rapid Amplification of cDNA Ends	p. 105
Randomly Amplified Polymorphic DNA Fingerprinting: The Basics	p. 117
Microsphere-Based Single Nucleotide Polymorphism Genotyping	p. 123
Ligase Chain Reaction	p. 135
Nested RT-PCR in a Single Closed Tube	p. 151
Direct PCR from Serum: Application to Viral Genome Detection	p. 161
Long PCR Amplification of Large Fragments of Viral Genomes: A Technical Overview	p. 167
Long PCR Methodology	p. 173
Ultrasensitive and Quantitative PCR
Qualitative and Quantitative PCR: A Technical Overview	p. 181
Ultrasensitive PCR Detection of Tumor Cells in Myeloma	p. 185
Ultrasensitive Quantitative PCR to Detect RNA Viruses	p. 197
Quantitative PCR for cAMP RI Alpha mRNA: Use of Site-Directed Mutation and PCR Mimics	p. 205
Quantitation of Multiple RNA Species	p. 211
Transcriptome Analysis
Differential Display: A Technical Overview	p. 217
AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs	p. 225
PCR Fluorescence Differential Display	p. 237
Microarray Analysis Using RNA Arbitrarily Primed PCR	p. 245
Oligonucleotide Arrays for Genotyping: Enzymatic Methods for Typing Single Nucleotide Polymorphisms and Short Tandem Repeats	p. 255
Serial Analysis of Gene Expression	p. 271
Mutations and Polymorphisms
Mutation and Polymorphism Detection: A Technical Overview	p. 287
Combining Multiplex and Touchdown PCR for Microsatellite Analysis	p. 295
Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluorescence-Based Multiplex Microsatellite PCR	p. 301
Reaction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA	p. 309
Degenerate Oligonucleotide-Primed PCR	p. 315
Mutation Detection Using RT-PCR-RFLP	p. 319
Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms	p. 323
PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA	p. 327
PCR-Based Sequencing
Sequencing: A Technical Overview	p. 337
Preparation and Direct Automated Cycle Sequencing of PCR Products	p. 341
Nonradioactive PCR Sequencing Using Digoxigenin	p. 347
Direct Sequencing by Thermal Asymmetric PCR	p. 355
Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing	p. 361
Direct Sequencing with Highly Degenerate and Inosine-Containing Primers	p. 367
Determination of Unknown Genomic Sequences Without Cloning	p. 373
Cloning PCR Products for Sequencing in M13 Vectors	p. 385
DNA Rescue by the Vectorette Method	p. 393
Technical Notes for Sequencing Difficult Templates	p. 401
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	p. 405
Cycling Primed In Situ Amplification	p. 425
Direct and Indirect In Situ PCR	p. 433
Reverse Transcriptase In Situ PCR: New Methods in Cellular Interrogation	p. 445
Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes	p. 453
Cloning and Mutagenesis
Cloning and Mutagenesis: A Technical Overview	p. 467
Using T4 DNA Polymerase to Generate Clonable PCR Products	p. 469
A T-Linker Strategy for Modification and Directional Cloning of PCR Products	p. 475
Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers	p. 485
cDNA Libraries from a Low Amount of Cells	p. 499
Creation of Chimeric Junctions, Deletions, and Insertions by PCR	p. 511
Recombination and Site-Directed Mutagenesis Using Recombination PCR	p. 517
Megaprimer PCR: Application in Mutagenesis and Gene Fusion	p. 525
Index	p. 533
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