By Jacqueline Boultwood
An extraordinary choice of all of the most modern recommendations for gene isolation and mapping, together with the newest equipment for gene characterization utilizing database analyses. This number of completely demonstrated recipes additionally contains chapters for the computational research of novel cDNA sequences with up to the moment info on uncomplicated series research, series similarity searches, exon detection and similarity searches, and the prediction of gene functionality. Its cutting-edge tools represent critical instruments for all scientists engaged within the look for particular sickness genes, or within the normal development of the human genome venture.
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Extra resources for Gene Isolation and Mapping Protocols
Example text
Autoradiography 1. Wrap the filter m plastic film, and placed in an X-ray film cassette. 2 Expose the filter to the film usually at -70°C for l-5 d to vrsuahze the hybridized fragments Develop the film usmg a commercial film developing system (see Notes 3 and 4). 2. Analysis of Hybrids Using PCR Amplification 1. Perform PCR m 50-yL reactions containing 30 ng template DNA, 1X PCR buffer, 20 pmol primer, 200 pA4 dNTPs, and 1 U of Tag polymerase on a thermocycler, for example, GeneAmp 9600 thermal cycler (Perkin-Elmer Cetus).
Large numbers of cells can be analyzedin a short time; 6. Data can be obtained from nondividing or terminally differentiated cells; and 7. The techniquehas beenadaptedfor automatedsystems 2. Several authors have addressed the apphcations of FISH m cytogenettc analysis, cancer diagnoses,and gene mapping (7,s). This chapter examines the role of FISH in gene mapping with an emphasis on detailed techniques. 1. Principles of FISH The technique of FISH is based on the same principle as Southern blot analysis, namely, the ability of single-stranded DNA to anneal to complementary DNA.
The disadvantages of this method were the relatively poor spatial resolution owing to scatter of the radioactive emissions, the length of time for the procedure (long autoradiographic exposure times were typically required), and the poor stability of the probes. The mtroduction of techniques to detect hybridized probes using fluorochromes in the late 1970s circumvented many of these problems (3,4); however, it was not until the end of the next decade that fluorescence in situ hybridization (FISH) techniques became widely applicable (5,6).