E-Book Overview
An outstanding collection of detailed, state-of-the-art techniques for studying many aspects of nervous system cell biology. The methods span a multidisciplinary range of cellular and molecular approaches to both normal and injured nervous system function, and particularly to neurodegenerative processes. Included are basic RT-PCR techniques, cell culture systems, second-messenger signaling methods, and patch-clamp techniques. These powerful techniques will illuminate nervous system function, injury, degeneration, and the repair/regenerative process.
E-Book Content
Introduction
to Polymerase
Chain Reaction
Mark C. Miller and Laura Cunningham 1. Introduction The polymerase cham reaction (PCR) has become an indispensable tool of molecular biology (I-5). Smce its discovery in 1985 the PCR process has found its integration into all research areas mvolving the use of DNA and RNA. Using this technique, a small starting sample of DNA or RNA can be used to amplify a specific DNA or RNA target over a million-fold in as little as 2 h. This allows for the detection of as little as a single copy of a gene or part of a gene m cells, whether they be from blood, cultured cells, tissue biopsies, chromosomes, or any other biological system that contains DNA or RNA, mcludmg archival materials (formalin-fixed, paraffin-embedded). Thus chapter will drscuss the prmciples of PCR as it relates to the methodologies employed to successfully carry out the reaction on DNA targets. Protocols relating to DNA extraction, PCR conditions, amplification cycles, and post-PCR detection methods will be presented. RNA amplification (RNA PCR) will be discussed in another chapter. There is a wide variety of methodologies for the extraction of DNA for use in many molecular-biology applications. When preparing DNA for use m PCR, the DNA extracteddoes not have to be of the highest quality.