PCR Cloning Protocols : From Molecular Cloning to Genetic Engineering / edited by Bruce A. White.

Format:

Book

Contributor:

White, Bruce A., editor.

SpringerLink (Online service)

Series:

Methods in molecular biology 1064-3745 ; 67.

Springer Protocols (Springer-12345)

Methods in Molecular Biology™, 1064-3745 ; 67

Language:

English

Subjects (All):

Life sciences.

Biochemistry.

Life Sciences.

Biochemistry, general.

Local Subjects:

Life Sciences.

Biochemistry, general.

Physical Description:

1 online resource (XIV, 490 pages).

Contained In:

Springer eBooks

Place of Publication:

Totowa, NJ : Humana Press : Imprint: Humana Press, 1997.

System Details:

text file PDF

Summary:

The advent of PCR, with its power to amplify tiny amounts of DNA, quickly spawned the development of many analytical procedures that are widely used for detection, measurement, and characterization. However, creative investigators soon discovered the power of PCR for synthetic or preparative uses. This volume focuses on such preparative PCR protocols, which can be used in the cloning and modification of DNA. PCR Cloning Protocols: From Molecular Cloning to Genetic Engineer- ing is divided into seven parts, each containing a collection of chapters address- ing a general approach or goal. Part I presents basic PCR protocols, emphasizing optimizing conditions for (he amplification of DNA fragments of several kilobases in length. Part 11 offers several procedures for cloning PCR prod- ucts, depending on whether a specific restriction site can be used in the clon- ing vector, the PCR product is to be gel purified before cloning, or the fragmeni needs to be inserted in one or both orientations. Part III includes several pro- tocols involved in the mutagenesis of DNA, either site-directed or not, as well as several approaches to recombinant PCR, either for mutagenesis or building a custom gene, as well as one chapter describing a specific use of in vitro selection. Part IV addresses the frequent need to amplify and clone segments of DNA that are to the right, left, or scattered within a stretch of DNA (either vector, chromosomal, or cDNA) of known sequence.

Contents:

Performing and Optimizing PCR

PCR

XL PCR Amplification of Long Targets From Genomic DNA

Amplification of DNA Sequences Up To 5 kb from Small Amounts of Genomic DNA Using Tub DNA Polymerase

One-Step Optimization Using Touchdown and Stepdown PCR

GC-Rich Template Amplification by Inverse PCR

Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments

Cloning PCR Products

Using T4 DNA Polymerase to Generate Clonable PCR Products

Rapid (Ligase-Free) Subcloning of PCR Products

Cloning PCR Products Utilizing the T/A Overhang and a Kit

Cloning Unmodified PCR Products Using Engineered Xcml Restriction Sites in a Portable Cassette

A T-Linker Strategy for Modification and Directional Cloning of PCR Products

Recovery of DNA Amplification Products from Silver-Stained Polyacrylamide Gels

Mutagenesis, Recombination, and In Vitro Selection

Recombination and Site-Directed Mutagenesis Using Recombination PCR

In Vitro Recombination and Mutagenesis of DNA

In-Frame Cloning of Synthetic Genes Using PCR Inserts

Creation of Chimeric Junctions, Deletions, and Insertions by PCR

Mutagenesis and Gene Fusion by Megaprimer PCR

Rapid and Efficient One-Tube PCR-Based Mutagenesis Method

Thermostable Ligase-Mediated Incorporation of Mutagenic Oligonucleotides During PCR Amplification

Linker Scanning Mutagenesis by Three-Step PCR

Sequence Inversion by Flip-PCR

PCR Site-Directed Mutagenesis Using Pyrococcus sp GB-D Polymerase Coupled to a Rapid Screening Procedure

Using the SELEX Combinatorial Chemistry Process to Find High Affinity Nucleic Acid Ligands to Target Molecules

Cloning Unknown Neighboring DNA

Rapid Amplification of cDNA Ends

Amplification of Gene-Regulating Regions with Single-Sided Specificity

An End-Trimming Method and Its Application to Amplify Adjacent cDNA and Genomic DNA Fragments by PCR

Anchoring a Defined Sequence to the 5? Ends of mRNAs

Rapid Directional Walk Within DNA Clones by Step-Out PCR

Inverse PCR

Rapid Amplification of Gene Ends (RAGE) from Gene Libraries by Anchored PCR

Isolation of Coding Sequences from Yeast Artificial Chromosome (Yac)

Library Construction and Screening

cDNA Libraries from a Low Amount of Cells

Rapid and Nonradioactive Screening of Recombinant Libraries by PCR

Use of PCR for cDNA Library Screening

Generation and PCR Screening of Bacteriophage ? Sublibraries Enriched for Rare Clones (the "Sublibrary Method")

Differential and Subtractive Approach By cDNA Analysis and Cloning

Normalization of cDNA Sequence Representation by Molecular Selection

Subtractive cDNA Cloning Using Magnetic Beads and PCR

Generation of a PCR-Renewable Source of Subtractive cDNA

The Use of PCR for Differential Screening of cDNA Libraries

Identification and Cloning of Differentially Expressed Genes by DDRT-PCR

Cloning Members of Gene Families

Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers

Amplification Using Degenerate Primers with Multiple Inosines to Isolate Genes with Minimal Sequence Similarity

Designing PCR Primers to Amplify Specific Members or Subgroups of Multigene Families

Screening Gene Family-Enriched cDNA Sublibraries with an Unamplified cDNA Probe.

Other Format:

Printed edition:

ISBN:

9781592595532

Access Restriction:

Restricted for use by site license.