PCR Cloning Protocols, Second Edition, updates and expands Bruce White's best-selling PCR Cloning Protocols (1997) with the newest procedures for DNA cloning and mutagenesis. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long distance PCR and GC-rich template amplification. Also included are both conventional and novel enzyme-free and restriction site-free procedures to clone PCR products into a range of vectors, as well as state-of-the-art protocols to facilitate DNA mutagenesis and recombination, and to clone the challenging uncharacterized DNA flanking a known DNA fragment.

Table of Contents:
Part I. Performing and Optimizing PCR Polymerase Chain Reaction: Basic Principles and Routine Practice Lori A. Kolmodin and David E. Birch Computer Programs for PCR Primer Design and Analysis Bing-Yuan Chen, Harry W. Janes, and Steve Chen Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming Kenneth H. Roux XL PCR Amplification of Long Targets from Genomic DNA Lori A. Kolmodin Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments Jyrki T. Aatsinki Long Distance Reverse-Transcription PCR Volker Thiel, Jens Herold, and Stuart G. Siddell Increasing PCR Sensitivity for Amplification from Paraffin-Embedded Tissues Abebe Akalu and Juergen K. V. Reichardt GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects Alain Moreau, Da Shen Wang, Steve Forget, Colette Duez, and Jean Dusart PCR Procedure for the Isolation of Trinucleotide Repeats Teruaki Tozaki Methylation-Specific PCR Haruhiko Ohashi Direct Cloning of Full-Length Cell Differentially Expressed Genes by Multiple Rounds of Subtractive Hybridization Based on Long-Distance PCR and Magnetic Beads Xin Huang, Zhenglong Yuan, and Xuetao Cao Part II. Cloning PCR Products Cloning PCR Products: An Overview Baotai Guo and Yuping Bi Using T4 DNA Polymerase to Generate Clonable PCR Products Kai Wang Enzyme-Free Cloning of PCR Products and Fusion Protein Expression Brett A. Neilan and Daniel Tillett Directional Restriction Site-Free Insertion of PCR Products into Vectors Guo Jun Chen Autosticky PCR: Directional Cloning of PCR Products with Preformed 5' Overhangs Jozsef Gal and Miklos Kalman A Rapid and Simple Procedure for Direct Cloning of PCR Products into Baculoviruses Tamara S. Gritsun, Michael V. Mikhailov, and Ernest A. Gould Part III. Mutagenesis and Recombination PCR Approaches to DNA Mutagenesis and Recombination: An Overview Binzhang Shen In-Frame Cloning of Synthetic Genes Using PCR Inserts James C. Pierce Megaprimer PCR Sailen Barik PCR-Mediated Recombination: A General Method Applied to Construct Chimeric Infectious Molecular Clones Guowei Fang, Barbara Weiser, Aloise Visosky, Timothy Moran, and Harold Burger PCR Method for Generating Multiple Mutations at Adjacent Sites Jiri Adamec A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes Franco Laccone PCR Screening in Signature-Tagged Mutagenesis of Essential Genes Dario E. Lehoux and Roger C. Levesque Staggered Extension Process (StEP) In Vitro Recombination Anna Marie Aguinaldo and Frances Arnold Random Mutagenesis by Whole-Plasmid PCR Amplification Donghak Kim and F. Peter Guengerich Part IV. Cloning Unknown Neighboring DNA PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants: An Overview Eric Ka-Wai Hui, Po-Ching Wang, and Szecheng J. Lo Long Distance Vectorette PCR (LDV PCR) James A. L. Fenton, Guy Pratt, and Gareth J. Morgan Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA ("Cold-Start Method") Michael Lardelli Inverse PCR: cDNA Cloning Sheng-He Huang Inverse PCR: Genomic DNA Cloning Ambrose Y. Jong, Anna T'ang, De-Pei Liu, and Sheng-He Huang Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers Sheng-He Huang, Hua-Yang Wu, and Ambrose Y. Jong The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR Vincent J. J. Martin and William W. Mohn Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis Martina Celerin and Kristin T. Chun Isolation of Large Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR Zhong-Nan Yang and T. Erik Mirkov A "Step Down" PCR-Based Technique for Walking Into and the Subsequent Direct Sequence Analysis of Flanking Genomic DNA Ziguo Zhang and Sarah Jane Gurr Part V. Library Construction and Screening Use of PCR in Library Screening: An Overview Jinbao Zhu Cloning of Homologous Genes by Gene-Capture PCR Renato Mastrangeli and Silvia Donini Rapid and Nonradioactive Screening of Recombinant Libraries by PCR Michael W. King Rapid cDNA Cloning by PCR Screening (RC-PCR) Toru Takumi Generation and PCR Screening of Bacteriophage l Sublibraries Enriched for Rare Clones (the "Sublibrary Method") Michael Lardelli PCR-Based Screening for Bacterial Artificial Chromosome Libraries Yuji Yasukochi A 384-Well Microtiter-Plate-Based Template Preparation and Sequencing Method Lei He and Kai Wang A Microtiter-Plate-Based High Throughput PCR Product Purification Method Ryan Smith and Kai Wang