In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe.
An in vivo 5'-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%-20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.
Kitazawa
S
S
2nd Department of Pathology, Kobe University, School of Medicine, Japan.
Takenaka
A
A
Abe
N
N
Maeda
S
S
Horio
M
M
Sugiyama
T
T
D001973:Bromodeoxyuridine; D002460:Cell Line; D004247:DNA; D015342:DNA Probes; D006801:Humans; D015151:Immunoblotting; D007938:Leukemia; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D009857:Oncogenes; D010957:Plasmids; D012313:RNA; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic
D015342:DNA Probes; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D001973:Bromodeoxyuridine