dUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase
| Autor: | V. A. Vasiliskov, Sergey Surzhikov, O. A. Zasedateleva, Igor P. Smirnov, Timur O. Guseinov, V. E. Kuznetsova, Alexander V. Chudinov, M. A. Spitsyn, V. E. Shershov, Roman A Yurasov |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Fluorophore Stereochemistry DNA-Directed DNA Polymerase Biology 010402 general chemistry 01 natural sciences Polymerase Chain Reaction Primer extension law.invention 03 medical and health sciences chemistry.chemical_compound Adenine nucleotide law Genetics Nucleotide Taq Polymerase Polymerase chain reaction Polymerase Fluorescent Dyes chemistry.chemical_classification Nucleotides fungi Templates Genetic Carbocyanines Nucleotidyltransferases 0104 chemical sciences Kinetics 030104 developmental biology chemistry biology.protein Methods Online Deoxyuracil Nucleotides DNA Taq polymerase |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3− and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3′ ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates. |
| Databáze: | OpenAIRE |
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