CHEMICALLY MODIFIED THERMOSTABLE ENZYME, METHOD OF ITS PREPARING, METHOD OF AMPLIFICATION OF NUCLEIC ACID IN SAMPLE, REACTION MIXTURE FOR ITS REALIZATION AND SET FOR CHAIN REACTION CARRYING OUT

摘要

biotechnology, molecular biology, nucleic acids, biochemistry. SUBSTANCE: invention relates to method of amplification of nucleic acid sequences. Chemically modified enzyme is characterized that thermostable DNA polymerase or thermostable ligase is inactivated reversibly in an aqueous buffer at alkaline pH value at temperature below about 25 C with the modifying agent that does not results to significant increase of enzyme activity for about less 20 min. Dicarboxylic acid anhydride is taken as the modifying agent. Incubation of chemically modified enzyme at temperature above about 50 C in an aqueous buffer pH value of which is brought about to 8-9 at 25 C results to at least two-fold increase of enzyme activity for about less 20 min. Chemically modified ligase or DNA polymerase is used for amplification of nucleic acids. For this purpose inactivated enzyme is reactivated by incubation of reaction mixture at increased temperature before amplification reaction carrying out or this process is a component of amplification reaction. Irreversibly inactivated thermostable enzyme used in polymerase chain reaction-amplification decreases nonspecific amplification significantly and increases amount of required amplified target-sequence significantly. EFFECT: improved method of enzyme preparing, enhanced effectiveness of amplification. 15 cl, 5 dwg, 10 ex