METHOD FOR PRODUCING LOW-MOLECULAR DNA WITH HIGH DEGREE OF PURITY Russian patent published in 2018 - IPC C07H1/08 C07H21/04 

FIELD: biotechnology.

SUBSTANCE: method for obtaining low molecular weight DNA from animal raw materials is proposed. Carry out destruction of cell membranes and proteins by alternating processes of frosting and defrosting of the homogenate in a regime that ensures the maximum complete destruction of cell membranes while maintaining the activity of intracellular enzymes. Frosting is carried out at a temperature of -40 to -50 °C, and subsequent defrosting at a temperature of 20 to 40 °C. Repeat frosting and defrosting cycles 3–5 times. Let homogenate to autolyze at a temperature of 20 to 40 °C for 8–24 hours up to the formation of a homogeneous viscous mass of gray color. Treat the autolyzate before its dissolution by a complex of neutral and alkaline exoproteases with a working temperature regime from 40 to 60 °C to proteolyze the residual proteins and their fragments. After deproteinization, separate the DNA aggregates with a neutral inert organic high molecular weight precipitant, such as polyethylene glycol with a molecular weight of 1 to 15 kDa or a poloxamer having a molecular weight of from 0.9 to 12.5 kDa in a concentration of not more than 30 % by weight, in the presence of sodium chloride to a concentration not exceeding 10 % by weight. Break DNA once by its sonication at a frequency of 70 to 100 kHz for no more than 9 minutes, allowing to obtain DNA fragments no longer than 700 bp.

EFFECT: method provides the yield of DNA from the raw material of more than 80 % of the original, the production of DNA fragments of 200–700 bp in length, decrease in the amount of impurities in the target product to 0,1 % or less.

9 cl, 1 dwg, 11 ex