Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B
The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo–) and Deep Vent (exo–) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chromophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.
Keywordsmodified nucleotides 2'-deoxycytidine-5'-triphosphate cyanine dyes substrate specificity DNA polymerases asymmetric PCR real-time PCR
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