5'-Dimethoxytrityl protection group of synthetic oligonucleotide stability in polymerase chain reaction

Modification of 5'-OH group of nucleic acids by substituents with various physicochemical properties is important for molecular biology. Investigation of symmetric and asymmetric substitution of 5'-hydroxyl of dsDNA by 4,4'-dimetho-xytrityl (DMT) group seems interesting. Symmetric substitution can allow performing a selective ligation of dsDNA assembled from oligonucleotides by polymerase chain reaction as compared to shortened assembly by-products into a plasmid vector. Synthesis of asymmetrically labeled 5'-DMT dsDNA in case of presence of exonuclease specific to 5'-end modifications will allow obtaining a long synthetic ssDNA used for site-specific gene insertion into a genome by CRISPR/Cas9 technique. To conduct such investigations, it is necessary to know whether synthetic 5'-DMT oligonucleotide is stable under PCR conditions. Here we demonstrated by high performance liquid chromatography with UV (260 nm) and mass-spectrometric detection that 5'-DMT group of synthetic oligonucleotide is stable under PCR conditions but the presence of thiol compounds can decrease a yield of 5'-DMT dsDNA. We plan a further research on influence of 5'-DMT group of synthetic DNA on functionality of various enzymes.

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