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In Situ Hybridization - Controlling The Specificity Of The Cytochemical Assays

dna detection rna sequence

Good cytochemists know that experimental results must be checked and verified. For this reason, in addition to testing for reactions with their target DNA or RNA, they also test for reactions with unrelated nucleotide sequences. Likewise, they test for reactions with other components of the detection systems.

There are a series of controls that are run that detect if the labeling pattern is due to the proper sequence of reactants. For example, if the cyto-chemist leaves out the probe in the hybridization solution, there should be no reaction. Similarly, if the cytochemist changes the sequence of the probe, or uses a noncomplementary probe, there should be no reaction (unless that new sequences reacts with another sequence in the cell). Tests of the detection system must also be run by leaving out one or more components to learn if the reaction is dependent totally on the complete sequence of reactants.

SEE ALSO DNA; NUCLEOTIDE; RNA.

Gwen V. Childs

Bibliography

Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.

Brahic, M., and A.T. Haase. "Detection of Viral Sequences of Low Reiteration Frequency by In situ Hybridization." Proceedings of the National Academy of Science USA 75 (1978): 6125-6127.

Buongiorno-Nardelli, S., and F. Amaldi. "Autoradiographic Detection of Molecular Hybrids between rRNA and DNA in Tissue Sections." Nature 225 (1970): 946-948.

Childs, G. V. "In situ Hybridization with Nonradioactive Probes." In Methods in Molecular Biology, vol. 123: In situ Hybridization Protocols, I. A. Darby, ed. Totowa, NJ: Humana Press, Inc, 1999.

Gall, J. G., and M. Pardue. "Formation and Detection of RNA-DNA Hybrid Molecules in Cytological Preparation." Proceedings of the National Academy of Science USA 63 (1969): 378-383.

Haase, A.T., P. Venture, C. Gibbs, and W. Touretellotte. "Measles Virus Nucleotide Sequences: Detection by Hybridization In situ." Science 212 (1981): 672-673.

John, H. A., M. L. Birnstiel, and K. W. Jones. "RNA-DNA Hybrids at the Cytological Level." Nature 223 (1969): 582-587.

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almost 5 years ago

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