Analysis of spontaneous and psoralen-induced Salmonella typhimurium hisG46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences.
An improved DNA colony-hybridization method for the rapid characterization of Salmonella typhimurium hisG46 revertants is described. Oligodeoxyribonucleotides (15-mers) complementary to each of 6 possible transition or transversion mutations and an extragenic suppressor mutation, underlying the His+ phenotype, were prepared. Optimal sequence discrimination was achieved by hybridizing 15-mers at the apparent dissociation temperature (Td) for 2 h with chromosomal DNA of revertant colonies affixed to Whatman 541 filters. Subsequent exposure of filters to UVA radiation (320-400 nm) in the presence of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) resulted in cross-linking of perfectly matched probes and target DNA sequences while sequences containing a single base-pair mismatch could be discriminated with a brief denaturing wash. No false negative results were obtained with the new procedure. An analysis of 204 spontaneous and 174 PUVA-induced TA100 revertants is presented.
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Division of Microbiology, Food and Drug Administration, Washington, DC 20204.
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D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics* / Q000528:radiation effects; D005363:Ficusin / Q000494:pharmacology*; D011564:Furocoumarins / Q000494:pharmacology*; D008722:Methods; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D015203:Reproducibility of Results; D012486:Salmonella typhimurium / Q000235:genetics*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D011564:Furocoumarins; D009838:Oligodeoxyribonucleotides; D005363:Ficusin