Open AccessStudy of the multiple incorporation of modified nucleotides into the growing DNA strand
Author(s) -
О. С. Волкова,
А. В. Чудинов,
S. A. Lapa
Publication year - 2021
Publication title -
tonkie himičeskie tehnologii
Language(s) - English
Resource type - Journals
eISSN - 2686-7575
pISSN - 2410-6593
DOI - 10.32362/2410-6593-2021-16-2-148-155
Subject(s) - dna polymerase , polymerase , primer (cosmetics) , nucleotide , chemistry , dna , biochemistry , primase , microbiology and biotechnology , stereochemistry , biology , polymerase chain reaction , reverse transcriptase , organic chemistry , gene
Objectives . This study investigated the substrate properties of the modified derivatives of triphosphates of purine and pyrimidine deoxynucleosides (5-propynyl-2’-deoxyuridine-5’-triphosphate, 5-propynyl2’-deoxycytidine-5’-triphosphate, 5-methyl-2’-deoxycytidine-5’-triphosphate, and N6-methyl-2’-deoxyadenosine-5’-triphosphate) during their simultaneous incorporation in enzymatic reactions (polymerase chain and primer extension reactions). Methods . The real-time polymerase chain and primer extension reactions were used to study the substrate efficiency of modified deoxynucleotide triphosphates. Various pairwise combinations of modified derivatives were used; specially designed synthetic DNA fragments and libraries for the Systematic Evolution of Ligands by Exponential Enrichment technology were used as templates. Reactions were conducted using DNA polymerases: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. Results . In each case, a pair of compounds (modified dUTP + dCTP, dUTP + dATP, and dCTP + dATP) was selected to study the simultaneous incorporation into the growing DNA strand. The most effective combinations of nucleotides for simultaneous insertion were dU and dC, having 5-propynyl substitution. The Vent (exo-) DNA polymerase was found as the most effective for the modified substrates. Conclusions . The selected compounds can be used for the enzymatic preparation of modified DNA, including aptamers with extended physicochemical properties.