Variety of the DNA Hybridization Rate and Its Relationship with High Order Structure of Single Stranded Nucleic Acids

The hybridization of oligo-DNAs complementary to the sequences of the genes for verotoxins (Shiga toxins) type 1 and 2 of enterohemorrhagic Escherichia coli (EHEC) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which had been optimized to obtain a high rate of hybridization. The time courses of fluorescence polarization for the fluorescently labeled oligomers (probe DNAs) mixed with the amplified DNA of the genes were recorded. The secondary structures of the amplified single-stranded DNAs were forecasted based on the calculation for minimum free energy at a specified minimum stacking length. Five probe DNA sequences were designed, some of which hybridized extremely rapidly with an amplified product for the gene of Shiga toxin type 1. In the cases using the two different probe DNAs, the hybridization was 90% complete in less than 1 min, considerably faster than that of the 3 min reported previously, while with another probe it was not complete in more than 14 min. The variety of the rate for hybridization could not be explained by melting temperature or G + C content of the probe sequences. It was suggested that the reason for the slow hybridization would be a steric hindrance, comparing the hybridization rates with the shapes around the binding sites for the probes in the secondary structures.