Detection of point mutation in bla T genes of Enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method.
Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.
Tham
T N
TN
Laboratoire des Sondes Froides, Institut Pasteur, Paris, France.
Guesdon
J L
JL
D000327:Adsorption; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D004755:Enterobacteriaceae / Q000235:genetics*; D005470:Fluorometry / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes