CLONING OF UNIVERSAL HYBRIDIZATION PROBES FOR TESTING MYCOPLASMAL CONTAMINATION IN CELL-CULTURES

Universal probes for detecting mycoplasmal rRNA among eukaryotic RNA w ere cloned from the ribosomal operon of Acholeplasma laidlawii. Before cloning, the thermodynamic parameters of duplex formation between hum an srRNA and DNA complementary to Mycoplasma capricolum srRNA were det ermined, to obviate cross-hybridization of the probes with eukaryotic rRNA. Such analysis revealed a region of mycoplasmal rDNA marked by hi gh free energy in duplex formation with mammalian srRNA and low free e nergy with prokaryotic srRNA. Three DNA fragments of different length containing this region were produced by PCR and cloned in pUC18. The r esulting probes were universal and highly specific, permitting detecti on of various mycoplasmal species. Blot hybridization of the probes wi th BsuRI digests of mycoplasmal DNA allowed species identification.