PCR Cloning Protocols (Methods in Molecular Biology)

✍ Scribed by Bing-Yuan Chen, Harry W. Janes

• Publisher: Humana Press
• Year: 2002
• Tongue: English
• Leaves: 433
• Series: Methods in Molecular Biology
• Edition: 2
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Preface......Page 5
Contents......Page 7
PERFORMING AND OPTIMIZING PCR......Page 15
Polymerase Chain Reaction......Page 16
Computer Programs for PCR Primer Design and Analysis......Page 32
Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming......Page 46
XL PCR Amplification of Long Targets from Genomic DNA......Page 52
Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments......Page 67
Long Distance Reverse-Transcription PCR......Page 73
Increasing PCR Sensitivity for Amplification from Paraffin-Embedded Tissues......Page 81
GC-Rich Template Amplification by Inverse PCR......Page 89
PCR Procedure for the Isolation of Trinucleotide Repeats......Page 95
Methylation-Specific PCR......Page 104
Direct Cloning of Full-Length Cell Differentially Expressed Genes by Multiple Rounds of Subtractive Hybridization Based on Long-Distance PCR and Magnetic Beads......Page 111
CLONING PCR PRODUCTS......Page 122
Cloning PCR Products......Page 123
Using T4 DNA Polymerase to Generate Clonable PCR Products......Page 132
Enzyme-Free Cloning of PCR Products and Fusion Protein Expression......Page 136
Directional Restriction Site-Free Insertion of PCR Products into Vectors......Page 144
Autosticky PCR......Page 151
A Rapid and Simple Procedure for Direct Cloning of PCR Products into Baculoviruses......Page 162
MUTAGENESIS AND RECOMBINATION......Page 173
PCR Approaches to DNA Mutagenesis and Recombination......Page 174
In-Frame Cloning of Synthetic Genes Using PCR Inserts......Page 182
Megaprimer PCR......Page 195
PCR-Mediated Recombination......Page 203
PCR Method for Generating Multiple Mutations at Adjacent Sites......Page 213
A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes......Page 223
PCR Screening in Signature-Tagged Mutagenesis of Essential Genes......Page 230
Staggered Extension Process (StEP) In Vitro Recombination......Page 240
Random Mutagenesis by Whole-Plasmid PCR Amplification......Page 245
CLONING UNKNOWN NEIGHBORING DNA......Page 250
PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants......Page 251
Long Distance Vectorette PCR (LDV PCR)......Page 277
Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA (“Cold-Start Method”)......Page 287
Inverse PCR......Page 294
Inverse PCR......Page 301
Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers......Page 308
The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR......Page 314
Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis......Page 323
Isolation of Large-Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR......Page 334
A “Step Down” PCR-Based Technique for Walking Into and the Subsequent Direct-Sequence Analysis of Flanking Genomic DNA......Page 340
LIBRARY CONSTRUCTION AND SCREENING......Page 348
Use of PCR in Library Screening......Page 349
Cloning of Homologous Genes by Gene-Capture PCR......Page 355
Rapid and Nonradioactive Screening of Recombinant Libraries by PCR......Page 373
Rapid cDNA Cloning by PCR Screening (RC-PCR)......Page 381
Generation and PCR Screening of Bacteriophage λ Sublibraries Enriched for Rare Clones (the “Sublibrary Method”)......Page 386
PCR-Based Screening for Bacterial Artificial Chromosome Libraries......Page 395
A 384-Well Microtiter-Plate-Based Template Preparation and Sequencing Method......Page 405
A Microtiter-Plate-Based High Throughput PCR Product Purification Method......Page 411
Index......Page 416