Book Info

Rutgers Univ., New Brunswick, NJ. Covers performing and optimizing PCR, cloning PCR products, mutagenesis and recombination, cloning unknown neighboring DNA, and library construction and screening. Features reproducible methods for isolating genes from all biological samples. Hardcover, comb-bound not yet available. --This text refers to the Hardcover edition.

Book Description

This completely revised second edition provides proven protocols for PCR cloning and mutagenesis. It builds on its best-selling predecessor to allow researchers of all levels to increase their success rate in their experiments. The editor has divided the book into five parts, beginning with performing and optimizing PCR to Cloning PCR products, through to mutagenesis and recombination. The final two parts cover the cloning of unknown neighboring DNA and DNA library construction and screening. Each part includes an overview that summarizes the current methods available and their underlying strategies, advantages, and disadvantages for the particular research. The techniques bring to both new and established researchers the power to apply PCR-based methodology to the cloning and modification of DNA, either through innovative protocols or by fostering individual creativity to modify and customize the protocols to best fit their own needs.

Preface
Contributors
1 Polymerase Chain Reaction: Basic Principles and Routine Practice 3
2 Computer Programs for PCR Primer Design and Analysis 19
3 Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming 31
4 XL PCR Amplification of Long Targets from Genomic DNA 37
5 Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments 53
6 Long Distance Reverse-Transcription PCR 59
7 Increasing PCR Sensitivity for Amplification from Paraffin-Embedded Tissues 67
8 GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects 75
9 PCR Procedure for the Isolation of Trinucleotide Repeats 81
10 Methylation-Specific PCR 91
11 Direct Cloning of Full-Length Cell Differentially Expressed Genes by Multiple Rounds of Subtractive Hybridization Based on Long-Distance PCR and Magnetic Beads 99
12 Cloning PCR Products: An Overview 111
13 Using T4 DNA Polymerase to Generate Clonable PCR Products 121
14 Enzyme-Free Cloning of PCR Products and Fusion Protein Expression 125
15 Directional Restriction Site-Free Insertion of PCR Products into Vectors 133
16 Autosticky PCR: Directional Cloning of PCR Products with Preformed 5' Overhangs 141
17 A Rapid and Simple Procedure for Direct Cloning of PCR Products into Baculoviruses 153
18 PCR Approaches to DNA Mutagenesis and Recombination: An Overview 167
19 In-Frame Cloning of Synthetic Genes Using PCR Inserts 175
20 Megaprimer PCR 189
21 PCR-Mediated Recombination: A General Method Applied to Construct Chimeric Infectious Molecular Clones 197
22 PCR Method for Generating Multiple Mutations at Adjacent Sites 207
23 A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes 217
24 PCR Screening in Signature-Tagged Mutagenesis of Essential Genes 225
25 Staggered Extension Process (StEP) In Vitro Recombination 235
26 Random Mutagenesis by Whole-Plasmid PCR Amplification 241
27 PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants: An Overview 249
28 Long Distance Vectorette PCR (LDV PCR) 275
29 Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA ("Cold-Start Method") 285
30 Inverse PCR: cDNA Cloning 293
31 Inverse PCR: Genomic DNA Cloning 301
32 Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers 309
33 The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR 315
34 Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis 325
35 Isolation of Large Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR 337
36 A "Step Down" PCR-Based Technique for Walking Into and the Subsequent Direct Sequence Analysis of Flanking Genomic DNA 343
37 Use of PCR in Library Screening: An Overview 353
38 Cloning of Homologous Genes by Gene-Capture PCR 359
39 Rapid and Nonradioactive Screening of Recombinant Libraries by PCR 377
40 Rapid cDNA Cloning by PCR Screening (RC-PCR) 385
41 Generation and PCR Screening of Bacteriophage [lambda] Sublibraries Enriched for Rare Clones (the "Sublibrary Method") 391
42 PCR-Based Screening for Bacterial Artificial Chromosome Libraries 401
43 A 384-Well Microtiter-Plate-Based Template Preparation and Sequencing Method 411
44 A Microtiter-Plate-Based High Throughput PCR Product Purification Method 417
Index 423