Distinguished scientists and researchers present a comprehensive collection of current preparative PCR techniques that can be used in cloning and modifying DNA and cDNA. Topics include performing and optimizing PCR (including long PCR), cloning PCR products, cloning unknown neighboring DNA, and library construction and screening. Also covered are mutagenesis, recombination, and in vitro selection, differential and subtractive approaches to cDNA analysis and screening, and cloning members of gene families. 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.
This volume provides protocols useful to both the inexperienced and the experienced investigator. Most importantly it highlights the emerging role of PCR as a versatile tool for the molecular biologist eliminating many laborious and expensive techniques associated with traditional gene isolation and analysis. - Biochemical Education
Part I. Performing and Optimizing PCR. PCR: Basic Principles and Routine Practice Lori A. Kolmodin and J. Fenton Williams. XL PCR Amplification of Long Targets from Genomic DNA Suzanne Cheng and Lori A. Kolmodin. Amplification of DNA Sequences Up to 5 kB from Small Amounts of Genomic DNA Using Tub DNA Polymerase Helen B. Forrester and Ian R. Radford. One-Step Optimization Using Touchdown and Stepdown PCR Kenneth H. Roux and Karl H. Hecker. GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects Alain Moreau Collete Duez and Jean Dusart. Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments Jyrki T. Aatsinki. Part II. Cloning PCR Products. Using T4 DNA Polymerase to Generate Clonable PCR Products Kai Wang. Rapid (Ligase-Free) Subcloning of PCR Products Alan R. Shuldiner and Keith Tanner. Cloning PCR Products Utilizing the T/A Overhang and a Kit Melissa Lail-Trecker. Cloning Unmodified PCR Products Using Engineered Xcml Restriction Sites in a Portable Cassette Alessandro Testori and Paul Sollitti. A T-Linker Strategy for Modification and Directional Cloning of PCR Products Robert M. Horton Raghavanpillai Raju and Bianca M. Conti-Fine. Recovery of DNA Amplification Products from Silver-Stained Polyacrylamide Gels: Applications in Nucleic Acid Fingerprinting and Genetic Mapping Gustavo Caetano-Annol's and Robert N. Trigiano. Part III. Mutagenesis Recombination and In Vitro Selection. Recombination and Site-Directed Mutagenesis Using Recombination PCR Douglas H. Jones and Stanley C. Winistorfer. In Vitro Recombination and Mutagenesis of DNA: SOEing Together Tailor-Made Genes Robert M. Horton. In-Frame Cloning of Synthetic Genes Using PCR Inserts James C. Pierce. Creation of Chimeric Junctions Deletions and Insertions by PCR GeneviSve Pont-Kingdon. Mutagenesis and Gene Fusion by Megaprimer PCR Sailen Barik. Rapid and Efficient One-Tube PCR-Based Mutagenesis Method Veronique Picard and Susan Clark Bock. Thermostable Ligase-Mediated Incorporation of Mutagenic Oligonucleotides During PCR Amplification Scott F. Michael. Linker Scanning Mutagenesis by Three-Step PCR Judith T. Schanke. Sequence Inversion by Flip-PCR Judith T. Schanke. PCR Site-Directed Mutagenesis Using Pyrococcus sp GBN-D Polymerase Coupled to a Rapid Screening Procedure: Application to a b- Glucanase Gene Jaume Pons Antoni Planas Miguel Juncosa and Enrique Querol. Using the SELEX Combinatorial Chemistry Process to Find High Affinity Nucleic Acid Ligands to Target Molecules Craig Tuerk. Part IV. Cloning Unknown Neighboring DNA. Rapid Amplification of cDNA Ends David Bertioli. Amplification of Gene-Regulating Regions with Single-Sided Specificity Mei-Zhong Luo and Rino Cella. An End-Trimming Method and Its Application to Amplify Adjacent cDNA and Genomic DNA Fragments by PCR Hiroyuki Iwahana and Mitsuo Itakura. Anchoring a Defined Sequence to the 5' Ends of mRNAs: The Bolt to Clone Rare Full-Length mRNAs Jean Baptiste Dumas Milne Edwards Olivier Valdenaire and Jacques Mallet. Rapid Directional Walk Within DNA Clones by Step-Out PCR Umadevi V. Wesley and Cedric S. Wesley. Inverse PCR: An Efficient Approach to Cloning cDNA Ends Sheng-He Huang. Rapid Amplification of Gene Ends (RAGE) from Gene Libraries by Anchored PCR Sheng-He Huang and Ambrose Y. Jong. Isolation of Coding Sequences from Yeast Artificial Chromosome (Yac): Clones by Exon Amplification Fernando Gibson and Steve D. M. Brown. Part V. Library Construction and Screening. cDNA Libraries from a Low Amount of Cells Phillippe Ravassard Christine Icard-Liepkalns Jacques Mallet and Jeane Baptiste Dumas Milne Edwards. Rapid and Nonradioactive Screening of Recombinant Libraries by PCR Michael W. King. Use of PCR for cDNA Library Screening Toru Takumi. Generation and PCR Screening of Bacteriophage l Sublibraries Enriched for Rare Clones (the Sublibrary Method) Michael Lardelli. Part VI. Differential and Subtractive Approach by cDNA Analysis and Cloning. Normalization of cDNA Sequence Representation by Molecular Selection Thierry G. Coche. Subtractive cDNA Cloning Using Magnetic Beads and PCR Thierry G. Coche. Generation of a PCR-Renewable Source of Subtractive DNA W. Michael Kuehl and James Battey. The Use of PCR for Differential Screening of cDNA Libraries Mark G. Thomas Sarah A. Hesse Yvonne J. Foss and Farzin Farzaneh. Identification and Cloning of Differentially Expressed Genes by DDRT-PCR Mikkel Rohde Rene Hummel Niels Pallisgaard Tino Podstufka Heidi Riedel Henrik Leffers and Michael Strauss. Part VII. Cloning Members of Gene Families. Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers Gregory M. Preston. Amplification Using Degenerate Primers with Multiple Inosines to Isolate Genes with Minimal Sequence Similarity Simona Bartl. Designing PCR Primers to Amplify Specific Members or Subgroups of Multigene Families Robert M. Horton Raghavanpillai Raju and Bianca M. Conti-Fine. Screening Gene Family-Enriched cDNA Sublibraries with an Unamplified cDNA Probe: Focusing on Moderately to Abundantly Expressed Clones Meimei Hu and Bruce A. White. Index.
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