Email this article Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR
- Authors: Lapa S.A.1, Pavlov A.S.1,2, Kuznetsova V.E.1, Shershov V.E.1, Spitsyn M.A.1,2, Guseinov T.O.1,2, Radko S.P.2,3, Zasedatelev A.S.1, Lisitsa A.V.3, Chudinov A.V.1,2
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Affiliations:
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
- OOO IBMC-EcoBioPharm
- Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
- Issue: Vol 53, No 3 (2019)
- Pages: 460-469
- Section: Structural and Functional Analysis of Biopolymers and Their Complexes
- URL: https://journals.rcsi.science/0026-8933/article/view/163986
- DOI: https://doi.org/10.1134/S0026893319030099
- ID: 163986
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Abstract
The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP–template–polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature–time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).
About the authors
S. A. Lapa
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Author for correspondence.
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991
A. S. Pavlov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991; Moscow, 119121
V. E. Kuznetsova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991
V. E. Shershov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991
M. A. Spitsyn
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991; Moscow, 119121
T. O. Guseinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991; Moscow, 119121
S. P. Radko
OOO IBMC-EcoBioPharm; Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
Email: lapa@biochip.ru
Russian Federation, Moscow, 119121; Moscow, 119121
A. S. Zasedatelev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991
A. V. Lisitsa
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
Email: lapa@biochip.ru
Russian Federation, Moscow, 119121
A. V. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
Russian Federation, Moscow, 119991; Moscow, 119121
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