The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization.
The hybridization of oligonucleotide sequences complementary to the genes of Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli (EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which was optimized to obtain a higher rate of hybridization. The time courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA in HCV, respectively. Probe DNA sequences were designed which hybridized extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and that of HCV. In the cases using the three different DNA probes, the hybridization was 90% complete in about 1 min, considerably faster than that of the 3 min reported previously. The rapidity of this hybridization could not be explained by the melting temperature or the G+C content of the probe sequences but its relationship with high order structure of the single stranded DNA or RNA of the amplicons in the solution was strongly suggested.
Tsuruoka
M
M
Advanced Science and Technology Laboratory, Hiroshima City, Ozuka 151-366 Hiroshima-shi, Hiroshima 731-3162, Japan. tsuruoka@sentan.city.hiroshima.jp
Murano
S
S
Okada
M
M
Ohiso
I
I
Fujii
T
T
D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D015342:DNA Probes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D005798:Genes, Bacterial; D005814:Genes, Viral; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics
D015342:DNA Probes; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2