PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells.
Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis, have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5 degrees C, precoated strips can still be used for hybridization up to at least 10 months.
Chevrier
D
D
Laboratoire de Prédéveloppement des Sondes, Institut Pasteur, Paris, France.
Rasmussen
S R
SR
Guesdon
J L
JL
D001483:Base Sequence; D015139:Blotting, Southern; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D014376:Tuberculosis / Q000175:diagnosis
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes