In-Situ PCR Techniques

ISBN-13: 9780471159469 / Angielski / Miękka / 1997 / 160 str.

Omar Bagasra; John Hansen

Opis

Protocols, tested and validated for their sensitivity, precision and reproducibility, for in-situ PCR detection localization of RNA and DNA are presented in this book. The authors give advice on the design of primers for PCR applications, and optimizing these protocols with plant, insect and prokayotic cells.

Spis treści

Overview;
Review of the PCR Technique;
Preliminary Solution–Based Reactions;
Preparation of Glass Slides and Tissues;
In Situ PCR: DNA and RNA Targets;
Special Applications of In Situ Amplification;
Hybridization Reactions;
Validation and Controls;
Materials and Methods;
Select Biography;
Appendices;
Index;
About the Authors.

Blurb

In situ gene amplification techniques offer tremendous potential as aids to clinical diagnosis through their ability to detect a single copy of a specific microbial, neoplastic, messenger, or mutated nucleic acid sequence in a cell smear, cell suspension, tissue section, or chromosome. In situ hybridization, applied in combination with the polymerase chain reaction (PCR), can be expected to fuel accelerated developments in the understanding of embryogenesis, organogenesis, and cell differentiation, as well as the pathogenesis of numerous disease processes. But the procedures are cumbersome and fraught with potential variables, and experimental results are difficult to reproduce.

In Situ PCR Techniques addresses this problem directly, with comprehensive step–by–step protocols for the delineation of genetic amplification and histological detection techniques. Each procedure has been tested and validated for its sensitivity, precision, and reproducibility, and the authors give advice on the design of primers for PCR applications and on optimizing these protocols for use with plant, insect, and prokaryotic cells. They facilitate the repetition of published experiments by providing the kinds of hints, tips, and laboratory secrets that are often left out of scientific papers. They also demonstrate clearly and thoroughly the efficacy of in situ PCR as an alternative to standard and fluorescence in situ hybridization for detecting a single copy of a mutant gene, a virus, or a very low–abundance message in individual cells, while preserving the morphology of the cell and the tissue in which it resides.

Topics covered include:
∗ A review of the in situ PCR technique
∗ Step–by–step protocols for in situ PCR techniques
∗ Optimization of annealing temperatures for specific primers
∗ Preparation of glass slides and tissues
∗ Selection of DNA and RNA targets

Supplemented with numerous helpful graphics and illustrations, In Situ PCR Techniques is immensely useful, not merely to the novice, but also to experienced researchers in investigative pathology, virology, and gene therapy, as well as in developmental biology, immunology, plant molecular biology, entomology, and the neurosciences.