Abstract
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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Abbreviations
- PCR:
-
polymerase chain reaction
- Cy5dUTP:
-
fluorescently labeled analogs of 2′-deoxyuridine 5′-triphosphate
- Cy5-dU:
-
modified nucleotides in DNA
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Original Russian Text © T.S. Lisitsa, V.E. Shershov, M.A. Spitsyn, T.O. Guseinov, A.Yu. Ikonnikova, D.O. Fesenko, S.A. Lapa, A.S. Zasedatelev, A.V. Chudinov, T.V. Nasedkina, 2017, published in Biofizika, 2017, Vol. 62, No. 3, pp. 464–471.
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Lisitsa, T.S., Shershov, V.E., Spitsyn, M.A. et al. The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides. BIOPHYSICS 62, 366–372 (2017). https://doi.org/10.1134/S0006350917030095
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DOI: https://doi.org/10.1134/S0006350917030095