2017  0,977
2016  0,799
2015  0,662
2014  0,740
2013  0,739
2012  0,637
2011  0,658
2010  0,654
2009  0,570
2008  0,849
2007  0,805
2006  0,330
2005  0,435
2004  0,623
2003  0,567
2002  0,641
2001  0,490
2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 52(2018) N 3 p. 458-466; DOI 10.1134/S0026893318030044 Full Text

D.O. Fesenko, T.O. Guseinov, S.A. Lapa, V.E. Kuznetsova, V.E. Shershov, M.A. Spitsyn, T.V. Nasedkina, A.S. Zasedatelev, A.V. Chudinov*

Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia

Received - 2017-06-06; Accepted - 2017-06-24

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 chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base.

modified nucleotides, 2'-deoxycytidine-5'-triphosphate, cyanine dyes, substrate specificity, DNA polymerases, asymmetric PCR, real-time PCR