Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain.
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.
Melloni
R H
RH
Molecular Neurobiology Laboratory, University of Massachusetts Medical Center, Worcester 01655, USA.
Aronin
N
N
DeGennaro
L J
LJ
Ferris
C F
CF
Harrison
R J
RJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D053938:DNA Fragmentation; D015342:DNA Probes; D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D000072159:Dynactin Complex; D006624:Hippocampus / Q000378:metabolism; D017403:In Situ Hybridization; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009496:Neurotensin / Q000235:genetics / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D017207:Rats, Sprague-Dawley; D016704:Synapsins / Q000235:genetics / Q000378:metabolism
D015342:DNA Probes; D018076:DNA, Complementary; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D016704:Synapsins; D009496:Neurotensin; C027908:endodeoxyribonuclease DdeI; D015252:Deoxyribonucleases, Type II Site-Specific