Abstract:
: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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Zasedateleva OA,Vasiliskov VA,Surzhikov SA,Kuznetsova VE,Shershov VE,Guseinov TO,Smirnov IP,Yurasov RA,Spitsyn MA,Chudinov AVdoi
10.1093/nar/gky247subject
Has Abstractpub_date
2018-07-06 00:00:00pages
e73issue
12eissn
0305-1048issn
1362-4962pii
4965821journal_volume
46pub_type
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