DNA-SEQUENCING BY HYBRIDIZATION WITH OLIGONUCLEOTIDE MATRIX (SHOM) - THEORY OF WASHING OUT THE DNA AFTER HYBRIDIZATION

A theory is put forward for washing out a single-stranded DNA fragment after hybridization with short oligonucleotides immobilized in a volu me of polyacrylamide gel. The theory adequately describes the main exp erimental facts we obtained earlier while developing a new method of D NA sequencing based on hybridization of the DNA fragment with a matrix of immobilized oligonucleotides Khrapko et al., J. DNA Seq. Mapp., 1 , 373-388 (1991)!. In particular, the theory explains and gives a sati sfactory quantitative description of the experimentally observed depen dence of the washing rate and the ''wash-off temperature'' on the conc entration of gel-immobilized oligonucleotides. Consideration is given to how the ''wash-off temperature'' is influenced by both the physicoc hemical parameters (enthalpy and entropy of duplex formation, and diff usion coefficient) and those chosen by the experimenter (wash duration , gel thickness, and some others). A simple analytical expression is d erived to calculate the ''wash-off temperature'' as dependent on these parameters, and the probable reasons for some discrepancies between t he calculated and the experimental washing curves are discussed.