Oligonucleotide derivatives in the hybridization analysis of nucleic acids: II. Isothermal signal amplification in DNA analysis by minisequencing

An isothermal amplification of a reporter signal during the analysis of the hybridization of nucleic acids was studied by limited probe extension (minisequencing). The intensity of the reporter signal was shown to increase due to the multiple enzymatic labeling of the probes during consecutive hybridization with one DNA template in both the homophase and heterophase assays using various detection methods: radioisotope or fluorescent labeling or enzyme-linked assay. The kinetic scheme of the process was proposed and the kinetic parameters for each step were evaluated. It was shown that the signal intensity correlated with the physicochemical characteristics for probe/DNA and product/DNA complexes. The maximum intensity was observed at the minimal difference between the thermodynamic stability of these complexes, provided that the reaction temperature was close to their melting temperature values; increasing or decreasing the reaction temperature led to a decrease in the amount of the reporting product. The signal intensity is significantly reduced when the analyzed DNA contains single-nucleotide discrepancies. The limited probe extension assay is useful not only for the detection of analyzed DNA, but also for its quantitative characterization.