电邮这篇文章 Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR
- 作者: 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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隶属关系:
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
- OOO IBMC-EcoBioPharm
- Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
- 期: 卷 53, 编号 3 (2019)
- 页面: 460-469
- 栏目: 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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详细
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).
作者简介
S. Lapa
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
编辑信件的主要联系方式.
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991
A. Pavlov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991; Moscow, 119121
V. Kuznetsova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991
V. Shershov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991
M. Spitsyn
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991; Moscow, 119121
T. Guseinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991; Moscow, 119121
S. Radko
OOO IBMC-EcoBioPharm; Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119121; Moscow, 119121
A. Zasedatelev
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991
A. Lisitsa
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Sciences
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119121
A. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences; OOO IBMC-EcoBioPharm
Email: lapa@biochip.ru
俄罗斯联邦, Moscow, 119991; Moscow, 119121
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