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Journal of Histochemistry and Cytochemistry, Vol. 45, 755-764, Copyright © 1997 by The Histochemical Society, Inc.

TECHNICAL NOTE

Dde-I Restriction Endonuclease Fragmentation: A Novel Method of Generating cDNA Probes for In Situ Hybridization in Brain

Richard H. Melloni, Jr.a, Neil Aroninb, Louis J. DeGennaroc, Craig F. Ferrisb, and Robert J. Harrisona
a Molecular Neurobiology Laboratory, University of Massachusetts Medical Center, Worcester, Massachusetts
b Program in Neuropsychiatric Sciences, Departments of Psychiatry and Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts
c Wyeth Ayerst Research, Princeton, New Jersey

Correspondence to: Richard H. Melloni, Jr., Molecular Neurobiology Lab., Neuropsychiatric Sciences Program, Dept. of Psychiatry, Univ. of Massachusetts Medical Ctr., 55 Lake Avenue N, Worcester, MA 01655.

We present a novel procedure for detection of low- and high-abundance messenger RNAs in the brain by in situ hybridization histochemistry, by using fragmented double-stranded cDNA as molecular probes. The procedure involves digesting the cDNA of interest with the restriction endonuclease from Desulfocibrio desulfuricans (Dde I digestion), followed by random primed labeling, which generates a family of high specific activity cDNA fragments. This procedure is a rapid, straightforward, and reproducible method of obtaining sensitive probes for in situ hybridization and is generally applicable to the analysis of the expression of a large number of genes. Here we report the use of this procedure to prepare probes for the detection of synapsin I, p150Glued, neurotensin, c-fos, and c-jun mRNAs in brain, using both isotopic and non-isotopic labeling methods. Because this procedure does not require complex recombinant DNA manipulations or oligonucleotide design, it should prove useful to the non-molecular biologist examining the expression of genes in the central nervous system. (J Histochem Cytochem 45:755-763, 1997)

Key Words: In situ hybridization, DNA probes, gene expression, mRNA


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