Study of Multiple Enzymatic Incorporation of Modified Nucleotides of Purine and Pyrimidine Nature in the Growing DNA Chain

The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction...

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Bibliographic Details
Published in:Molecular biology (New York) 2022, Vol.56 (1), p.115-123
Main Authors: Lapa, S. A., Volkova, O. S., Kuznetsova, V. E., Zasedatelev, A. S., Chudinov, A. V.
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
Deoxyribonucleic acid
DNA
DNA-directed DNA polymerase
Exonuclease
Human Genetics
Life Sciences
Nucleotides
Structural–Functional Analysis of Biopolymers and Their Complexes
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Summary:The substrate properties of nitrogen-base modified derivatives of purine and pyrimidine deoxynucleoside triphosphates during their simultaneous pairwise insertion into the growing DNA strand have been studied. Modified nucleotides were introduced using real-time PCR and the primer extension reaction; in one reaction, derivatives with both different and similar functional substituents were used. Genomic bacterial DNA, specially constructed synthetic DNA fragments, and SELEX libraries were used as templates. The reactions were performed using DNA polymerases with no 3'–5' correcting exonuclease activity: Taq, Vent (exo-), DeepVent (exo-), and KOD XL. It was shown that the substrate efficiency is affected by both the size of the substituent group and the chemical nature of deoxynucleoside triphosphate. The effectiveness varies significantly depending on the polymerase used. The most effective of the studied substrates are pyrimidine deoxynucleoside triphosphates in combination with Vent (exo-) DNA polymerase. DNAs modified by pairs of dissimilar nucleotides (dU + dC, dU + dA, dC + dA) with similar and different functional substituents were obtained.
ISSN:0026-8933
1608-3245
DOI:10.1134/S0026893322010046