PCR Cloning Protocols / Edition 2

by Bing-Yuan Chen, Harry W. Janes

Overview

PCR Cloning Prools, Second Edition, updates and expands Bruce White's best-selling PCR Cloning Prools (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 prools to facilitate DNA mutagenesis and recombination, and to clone the challenging uncharacterized DNA flanking a known DNA fragment.

Product Details

ISBN-13:	9780896039698
Publisher:	Springer-Verlag New York, LLC
Publication date:	04/01/2002
Series:	Methods in Molecular Biology , #192
Edition description:	2nd ed. 2002
Pages:	439
Product dimensions:	6.10(w) x 9.25(h) x 0.04(d)

About the Author

Chen, Bing-Yuan (Rutgers University, New Brunswick, NJ)
Janes, Harry W. (Rutgers University, New Brunswick, NJ)

Table of Contents

Performing and Optimizing PCR.- Polymerase Chain Reaction.- Computer Programs for PCR Primer Design and Analysis.- Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming.- XL PCR Amplification of Long Targets from Genomic DNA.- Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments.- Long Distance Reverse-Transcription PCR.- Increasing PCR Sensitivity for Amplification from Paraffin-Embedded Tissues.- GC-Rich Template Amplification by Inverse PCR.- PCR Procedure for the Isolation of Trinucleotide Repeats.- Methylation-Specific PCR.- Direct Cloning of Full-Length Cell Differentially Expressed Genes by Multiple Rounds of Subtractive Hybridization Based on Long-Distance PCR and Magnetic Beads.- Cloning PCR Products.- Cloning PCR Products.- Using T4 DNA Polymerase to Generate Clonable PCR Products.- Enzyme-Free Cloning of PCR Products and Fusion Protein Expression.- Directional Restriction Site-Free Insertion of PCR Products into Vectors.- Autosticky PCR.- A Rapid and Simple Procedure for Direct Cloning of PCR Products into Baculoviruses.- Mutagenesis and Recombination.- PCR Approaches to DNA Mutagenesis and Recombination.- In-Frame Cloning of Synthetic Genes Using PCR Inserts.- Megaprimer PCR.- PCR-Mediated Recombination.- PCR Method for Generating Multiple Mutations at Adjacent Sites.- A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing Repeat Expansions into CAG-Repeat Containing Genes.- PCR Screening in Signature-Tagged Mutagenesis of Essential Genes.- Staggered Extension Process (StEP) In Vitro Recombination.- Random Mutagenesis by Whole-Plasmid PCR Amplification.- Cloning Unknown Neighboring DNA.- PCR-Based Strategies to Clone Unknown DNA Regions from Known Foreign Integrants.-Long Distance Vectorette PCR (LDV PCR).- Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA (“Cold-Start Method”).- Inverse PCR.- 31 Inverse PCR.- Gene Cloning and Expression Profiling by Rapid Amplification of Gene Inserts with Universal Vector Primers.- The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions by Inverse PCR.- Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis.- Isolation of Large-Terminal Sequences of BAC Inserts Based on Double-Restriction-Enzyme Digestion Followed by Anchored PCR.- A #x201C;Step Down#x201D; PCR-Based Technique for Walking Into and the Subsequent Direct-Sequence Analysis of Flanking Genomic DNA.- Library Construction and Screening.- Use of PCR in Library Screening.- Cloning of Homologous Genes by Gene-Capture PCR.- Rapid and Nonradioactive Screening of Recombinant Libraries by PCR.- Rapid cDNA Cloning by PCR Screening (RC-PCR).- Generation and PCR Screening of Bacteriophage— Sublibraries Enriched for Rare Clones (the “Sublibrary Method ”).- PCR-Based Screening for Bacterial Artificial Chromosome Libraries.- A 384-Well Microtiter-Plate-Based Template Preparation and Sequencing Method.- A Microtiter-Plate-Based High Throughput PCR Product Purification Method.

Editorial Reviews

"This hardcover book is packed full of detailed prools and methodological articles on various aspects of PCR. The fact that the book was taken out of my hands by a colleague, to read up on touch-down PCR, as soon as I received it for review, attest to the book's usefulness. I will have to chain this book to my bookshelf, to make sure it does not disappear quickly-or I may consider buying a couple for the laboratory." -Plant Science

Review of the first edition.

This volume provides prools 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

From the Publisher

Researchers from a wide range of both countries and medical and biological disciplines compile proven protocols for cloning and mutagenesis using protein chain reaction (PCR) methods. They cover performing and optimizing PCR, cloning PCR products, mutagenesis and recombination, cloning unknown neighboring DNA, and constructing and screening libraries. The plastic comb binding lies open on the laboratory bench. The first edition was published in 1997 as . Annotation c. Book News, Inc., Portland, OR (booknews.com)

Review of the first edition. 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)

Biochemical Education

Reviewer: Eugene A Davidson, PhD (Georgetown University School of Medicine)
Description: This is a compilation of protocols involving the use of PCR. Publication of this second edition is appropriate since the first one appeared five years ago and many new techniques and applications have been developed in the interval.
Purpose: The intention of the book is to provide a comprehensive set of protocols covering all aspects of PCR. Given the wide use of this technology and its many applications, this is a worthwhile effort. The comprehensive and detailed protocols achieve this goal.
Audience: This is a laboratory manual intended for use by researchers — graduate students and post-doctoral fellows. There is enough detail so that new investigators should have little trouble executing experiments using these methods. The many authors are representative of active investigators in the field.
Features: There are 44 protocols encompassing virtually all aspects of PCR use. As one of the premier techniques of molecular biology, this is a useful addition to the literature. The book is divided into five sections. The initial material covers basic PCR techniques, primer design and amplification of GC-rich templates. This section is followed by one on cloning, protocols dealing with mutagenesis and then a section on cloning of unknown DNA adjacent to a sequence of interest. The last part deals with library construction. The utility of the book is enhanced by the inclusion, in each section, of an introductory chapter that provides an overview of the material to be covered. The protocols themselves are detailed enough to allow investigators unfamiliar with the particular technique to use it with confidence. Each section has a suitable bibliography. Laboratories using PCR will want to acquire this book. It should be noted that non-PCR DNA amplification methods are available and, in some cases, represent desirable alternatives.
Assessment: I will use this in my own laboratory since PCR still represents the method of choice for many applications, the protocols are detailed, and the directions are clear. We are, however, examining alternate methods.

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