METHOD FOR DETERMINING SUBSTRATE COMPATIBILITY OF MODIFIED DEOXYURIDINE TRIPHOSPHATE AND DNA POLYMERASE BY COMBINING QUANTITATIVE PCR AND ELECTROPHORESIS METHODS Russian patent published in 2020 - IPC C12Q1/68 

FIELD: chemistry; biotechnology; medicine.

SUBSTANCE: invention relates to molecular biology, organic chemistry, biotechnology, microbiology and medicine. Disclosed is a method for determining substrate compatibility of modified triphosphate deoxyuridine and DNA polymerase by combination by quantitative PCR and electrophoresis. Essence of the invention is that compatibility of modified nucleoside triphosphates and DNA polymerase is determined by a combination of methods: quantitative PCR in real time on a matrix of "conditionally double-stranded" DNA, composed of two synthetic oligonucleotides with a degenerate central part with mutually complementary flanking parts with complete replacement of the natural nucleotide with the modified analogue; quantitative PCR in real time on a bacterial DNA matrix, which is a PCR-product of a rpoB gene fragment with length of 215 base pairs with amplification of "enclosed" short internal fragment with length of 126 base pairs with complete replacement of natural nucleotide with modified analogue; electrophoretic control of PCR products with quantitative evaluation of full-size product. Modified 2'-deoxyuridine-5'-triphosphates contain in their structure functional groups, which are attached to 5-position of pyrimidine base with carbonylaminoallyl linker, allyl part of linker is connected with 5-position of pyrimidine base with trans-alkene bond. Functional groups are represented by linear aliphatic, branched aliphatic, phenylalkyl, alkylindole groups. DNA polymerase with absent correcting 3'-5'-exonuclease activity related to different families - Taq (family A) and Vent (exo-) (family B) was used. PCR was used with real-time registration, (Real time PCR) by accumulation of fluorescent signal from intercalating dye Eva Green (Biotium, USA) with excitation of fluorescence at wave length of about 500 nm and registration of about 530 nm. PCR products are separated by electrophoresis in 4 % agarose gel. Visualization of reaction products is carried out by agarose gel staining, in which PRC products are separated with ethidium bromide. Full-size product is quantitatively evaluated by optical density of the corresponding strips in the gel tracks. Proposed invention can be used in producing modified DNA fragments and modified aptamers, functional analogues of monoclonal antibodies.

EFFECT: invention provides determining substrate compatibility of nucleoside triphosphate derivatives and matrix-dependent DNA polymerases for producing high-efficiency modified DNA aptamers by SELEX method.

8 cl, 1 tbl, 104 ex, 3 dwg