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.
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