Gene Cloning and Analysis By RT-PCR

by Paul D. Siebert

This new molecular biology manual is designed to provide investigators with useful, up-to-date reverse transcription PCR-based methods for gene cloning and analysis. It contains detailed laboratory protocols in 24 original chapters prepared under the editorial direction of two of the pioneers of this technique by an international collaboration of 55 scientists noted for bringing new applications, improvements and fresh perspectives to the RT-PCR method. Since RT-PCR was first described for the detection of low-abundance mRNAs, the method has been extended to provide measurements of relative and absolute levels of single and multiple mRNAs. Methods for obtaining RNA for RT-PCR have also been extended to include analysis of mRNA levels in clinical samples and even in single cells.PCR methods are available for obtaining full-length cDNA, homologous cDNAs, and differentially expressed cDNAs. In many cases RT-PCR is able to circumvent many time-consuming and technically demanding cloning steps. The use of PCR can even enable cloning of DNA molecules without the need for biological hosts.The book is organized into five sections. The first reviews different methods for obtaining RNA for RT-PCR. Section II presents several new approaches for performing quantitative RT-PCR. The third section examines several PCR-based methods for cloning differentially expressed genes with descriptions of improved methods for performing differential RNA display and methods for PCR-based cDNA subtraction. Section IV recounts three PCR-based methods for obtaining full-length and homologous cDNAs. Finally, three ancillary RT-PCR methods are detailed in Section V.This volume is the first in an evolving series of molecular laboratory methods manuals from the publisher of BioTechniques Journal.

Spiral-bound

First published December 1, 1998