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
Medical Subject Headings
Animals; Brain; DNA Fragmentation; *DNA Probes; DNA, Complementary; Deoxyribonucleases, Type II Site-Specific; Hippocampus; *In Situ Hybridization; Microtubule-Associated Proteins; Neurotensin; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Rats; Rats, Sprague-Dawley; Synapsins
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.
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Citation: J Histochem Cytochem. 1997 May;45(5):755-63.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
Melloni, Richard H.; Aronin, Neil; DeGennaro, Louis J.; Ferris, Craig F.; and Harrison, Robert J., "Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain" (1997). GSBS Student Publications. 851.