[Study of the kinetics of the reaction reuniting DNA fragments catalyzed by DNA-ligase. Case of homogeneous molecules].

The kinetics of different DNA forms formation were studied during the ligation reaction. Reaction was carried out using as a model BamH1 DNA fragments of plasmid pBR322 and DNA-ligase of bacterophage T4. The products of the reaction were separated with 1% agarose gel, followed by DNA quantitation in the corresponding bands by scanning fluorimeter. The kinetics of the ligation reaction were measured in the range of DNA concentrations from 1 to 100 micrograms/ml. The system of differential equations, that described the kinetics of the ligation reaction, was obtained. This has no analytical solution, but may be approximately calculated with a computer. The analysis of kinetic equations was made in order to optimize the ligation reaction. The optimal DNA concentrations were found allowing the maximal yield of multimer circular DNA forms. The simple formula for calculatng these concentrations were developed. The yield of circular DNA multimers might be increased if the reaction is carried out at two stages: initially at high DNA concentration for linear multimers formation followed then a lower concentration after appropriated dilution. The formula for determiantion of the dilution time is presented. The data obtained allow to optimize the reaction conditions providing the recombinant DNA molecules formation.