Chemiprobe, a nonradioactive system for labeling nucleic acid. Principles and applications.
The Chemiprobe Kit provides a complete system for nonradioactive labeling of DNA probes and their detection in hybridization studies. The system is highly sensitive, permitting the detection of 0.2-0.4 pg DNA which allows detection of a single gene sequence in 0.5-1 microgram of bacterial DNA or in 3-5 micrograms of mammalian DNA. In this paper the authors show that the rRNA genes of M. capricolum can be detected by using only 50 ng/ml of sulfonated probe cloned from another mycoplasma, M. pneumoniae. The Chemiprobe system has been successfully used in the detection of the single copy human gene for glucocerobrosidase from total embryonic DNA by hybridization to a specific sulfonated cDNA. 5 x 10(4) M. pneumoniae cells can be detected either free or mixed with sputum using a standard dot blot technique: mycoplasma cells were lysed by a mucolytic agent, denaturated by NaOH, immobilized on a nylon membrane filter, and then hybridized with pPN4, a plasmid DNA probe specific for M. pneumoniae. The resulting hybrids were then detected by the standard Chemiprobe procedure. A new kit based on the Chemiprobe system has been designed especially for the detection of mycoplasmas in tissue culture. This kit has been tested on 70 random samples collected from tissue culture fluids from 11 different sources. Of these, 42 were found to be contaminated by the Chemiprobe procedure, whereas 41 were found to be contaminated by classical microbiological methods. No false negatives were found.
Nur
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Orgenics Ltd., Yavneh, Israël.
Reinhartz
A
A
Hyman
H C
HC
Razin
S
S
Herzberg
M
M
D004247:DNA; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008828:Microbiological Techniques; D009177:Mycoplasma pneumoniae; D009693:Nucleic Acid Hybridization / Q000502:physiology*; D010957:Plasmids; D013183:Sputum / Q000382:microbiology*
D004247:DNA