PCR Protocols By John M. S. Bartlett

Book Description:

In this new edition, the editors 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 proven 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. 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 trouble shooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results.

Table of contents :

Contents……Page 2
A Short History of the Polymerase Chain Reaction……Page 6
PCR Patent Issues……Page 10
Equipping and Establishing a PCR Laboratory……Page 18
Quality Control in PCR……Page 24
Extraction of Nucleic Acid Templates……Page 28
Extraction of DNA from Whole Blood……Page 30
DNA Extraction from Tissue……Page 34
Extraction of DNA from Microdissected Archival Tissues……Page 36
RNA Extraction from Blood……Page 44
RNA Extraction from Frozen Tissue……Page 46
RNA Extraction from Tissue Sections……Page 48
Dual DNA/RNA Extraction……Page 50
DNA Extraction from Fungi, Yeast, and Bacteria……Page 54
Isolation of RNA Viruses from Biological Materials……Page 56
Extraction of Ancient DNA……Page 58
DNA Extraction from Plasma and Serum……Page 64
Technical Notes for the Detection of Nucleic Acids……Page 66
Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels……Page 78
PCR Primer Design……Page 80
Optimization of Polymerase Chain Reactions……Page 88
Subcycling PCR for Long-Distance Amplifications of Regions with High and Low Guanine–Cystine Content……Page 100
Rapid Amplification of cDNA Ends……Page 104
Randomly Amplified Polymorphic DNA Fingerprinting……Page 116
Microsphere-Based Single Nucleotide Polymorphism Genotyping……Page 122
Ligase Chain Reaction……Page 134
Nested RT-PCR in a Single Closed Tube……Page 150
Direct PCR from Serum……Page 160
Long PCR Amplification of Large Fragments of Viral Genomes……Page 166
Long PCR Methodology……Page 172
Qualitative and Quantitative PCR……Page 178
Ultrasensitive PCR Detection of Tumor Cells in Myeloma……Page 182
Ultrasensitive Quantitative PCR to Detect RNA Viruses……Page 194
Quantitative PCR for cAMP RI Alpha mRNA……Page 202
Quantitation of Multiple RNA Species……Page 208
Differential Display……Page 212
AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs……Page 220
PCR Fluorescence Differential Display……Page 232
Microarray Analysis Using RNA Arbitrarily Primed PCR……Page 240
Oligonucleotide Arrays for Genotyping……Page 250
Serial Analysis of Gene Expression……Page 266
Mutation and Polymorphism Detection……Page 280
Combining Multiplex and Touchdown PCR for Microsatellite Analysis……Page 288
Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluorescence-Based Multiplex Microsatellite PCR……Page 294
Reduction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA……Page 302
Degenerate Oligonucleotide-Primed PCR……Page 308
Mutation Detection Using RT-PCR-RFLP……Page 312
Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms……Page 316
PCR-SSCP Analysis of Polymorphism……Page 320
Sequencing: A Technical Overview……Page 328
Preparation and Direct Automated Cycle Sequencing of PCR Products……Page 332
Nonradioactive PCR Sequencing Using Digoxigenin……Page 338
Direct Sequencing by Thermal Asymmetric PCR……Page 346
Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing……Page 352
Direct Sequencing with Highly Degenerate and Inosine-Containing Primers……Page 358
Determination of Unknown Genomic Sequences Without Cloning……Page 364
Cloning PCR Products for Sequencing in M13 Vectors……Page 376
DNA Rescue by the Vectorette Method……Page 384
Technical Notes for Sequencing Difficult Templates……Page 392
PCR-Based Detection of Nucleic Acids in Chromosomes, Cells, and Tissues……Page 394
Cycling Primed In Situ Amplification……Page 414
Direct and Indirect In Situ PCR……Page 422
Reverse Transcriptase In Situ PCR……Page 434
Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes……Page 442
Cloning and Mutagenesis……Page 454
A T-Linker Strategy for Modification and Directional Cloning of PCR Products……Page 462
Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers……Page 472
cDNA Libraries from a Low Amount of Cells……Page 486
Creation of Chimeric Junctions, Deletions, and Insertions by PCR……Page 498
Recombination and Site-Directed Mutagenesis Using Recombination PCR……Page 504
Megaprimer PCR……Page 512

PCR Protocols PDF

Author(s): John M. S. Bartlett, David Stirling (auth.), John M. S. Bartlett, David Stirling (eds.)

Series: Methods in Molecular Biology 226

Publisher: Humana Press, Year: 2003

ISBN: 9780896036277