THEORETICAL-ANALYSIS OF THE KINETICS OF DNA HYBRIDIZATION WITH GEL-IMMOBILIZED OLIGONUCLEOTIDES

A new method of DNA sequencing by hybridization using a microchip cont aining a set of immobilized oligonucleotides is being developed. A the oretical analysis is presented of the kinetics of DNA hybridization wi th deoxynucleotide molecules chemically tethered in a polyacrylamide g el layer. The analysis has shown that long-term evolution of the spati al distribution and of the amount of DNA bound in a hybridization cell is governed by ''retarded diffusion,'' i.e., diffusion of the DNA int errupted by repeated association and dissociation with immobile oligon ucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the st ate with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the char acteristic lime of retarded diffusion (which is proportional to the eq uilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. T his conclusion is indirectly confirmed by the observation of nonunifor m fluorescence of labeled DNA in perfect-match hybridization cells (br ighter at the edges), For optimal discrimination of perfect duplexes f rom duplexes with mismatches the hybridization process should be broug ht to equilibrium under low-temperature nonsaturation conditions for a ll cells, The kinetic differences between perfect and nonperfect duple xes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.