Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain
Graduate School of Biomedical Sciences; Department of Neurology and Cell Biology
Life Sciences | Medicine and Health Sciences
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.
DOI of Published Version
J Histochem Cytochem. 1997 May;45(5):755-63.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
Melloni RH, Aronin N, DeGennaro LJ, Ferris CF, Harrison RJ. (1997). Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain. Morningside Graduate School of Biomedical Sciences Student Publications. https://doi.org/10.1177/002215549704500514. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/851