The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides
- Авторы: Lisitsa T.S.1, Shershov V.E.1, Spitsyn M.A.1, Guseinov T.O.1, Ikonnikova A.Y.1, Fesenko D.O.1, Lapa S.A.1, Zasedatelev A.S.1, Chudinov A.V.1, Nasedkina T.V.1
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Учреждения:
- Engelhardt Institute of Molecular Biology
- Выпуск: Том 62, № 3 (2017)
- Страницы: 366-372
- Раздел: Molecular Biophysics
- URL: https://ogarev-online.ru/0006-3509/article/view/152299
- DOI: https://doi.org/10.1134/S0006350917030095
- ID: 152299
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Аннотация
The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.
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Об авторах
T. Lisitsa
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
V. Shershov
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
M. Spitsyn
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
T. Guseinov
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
A. Ikonnikova
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
D. Fesenko
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
S. Lapa
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
A. Zasedatelev
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
A. Chudinov
Engelhardt Institute of Molecular Biology
Email: nased@biochip.ru
Россия, Moscow, 119991
T. Nasedkina
Engelhardt Institute of Molecular Biology
Автор, ответственный за переписку.
Email: nased@biochip.ru
Россия, Moscow, 119991
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