Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis.
The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.
Kido
C
C
Advanced Science and Technology Laboratory, Hiroshima city, Ozuka 151-366, Asaminami-ku, Hiroshima 731-3162, Japan.
Murano
S
S
Tsuruoka
M
M
D015139:Blotting, Southern / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics
D004269:DNA, Bacterial; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2