E-Book Overview
This is a significant collection of established and novel methods for the successful quantitation of nucleic acids. Each method has been refined and tested by its developer and proven to work in such problems as the analysis of eukaryotic gene expression, the quantitation of viral loads in clinical specimens, reporter gene expression, and quantitative oncogene analysis. Particular emphasis is placed on the underlying principles of the design of competitive or noncompetitive standards, as well as on the optimization of the amplification process. In important cases several methods are given for the same problem so that readers may set up test systems tailored to their specific practical needs. With its step-by-step instructions, Quantitative PCR Protocols allows researchers to address biological and diagnostic questions that are difficult or impossible to answer using any other experimental approach.
E-Book Content
1 Quantitative
PCR
A Survey of the Present Technology Udo Reischl and Bernd Kochanowski 1. Introduction The polymerase chain reaction (PCR) IS a powerful tool for the amphficanon of trace amounts of nucleic acids, and has rapidly become an essential analytical tool for virtually all aspects of biological research in experrmental biology and medicine. Because the apphcatton of this technique provides unprecedented sensittvtty, it has facilitated the development of a variety of nucleic acid-based systems for diagnostic purposes, such as the detectton of viral (1) or bacterial pathogens (2), as well as genetic disorders (3), cancer (4J, and forensic analysis (5). These recently developed systemsopen up the possibihty of performing reliable diagnosis even before any symptoms of the disease appear, thus constderably improving the chances of success with treatment For many routme appltcattons, particularly in the diagnoses of viral mfecttons, the requir