A Simultaneous Use of Cy5-Modified Derivatives of Deoxyuridine and Deoxycytidine in PCR

Abstract

Particular features of the simultaneous incorporation into the growing DNA chain of modified Cy5-deoxyuridines (Cy5-dU) and deoxycytidines (Cy5-dC) and Taq DNA polymerase in the process of PCR amplification were studied. The studies were carried out for the pairwise incorporation of nucleotides modified with fluorophores charged positively, negatively, or neutral. A portion of each of the modified dNTP (Cy5-dUTP and Cy5-dCTP) in respect to natural analogs (dTTP and dCTP) was varied from 0 to 100%, i.e., until the complete substitution. The results were compared with the individual incorporation of the corresponding modified derivatives. The (Cy5-dU + Cy5-dC) pairs were used to amplify of the bacterial genome fragments of 126, 283, and 370 bp in length. Under the amplification conditions with Cy5-modified nucleotides the yield of the product decreased with an increase in the length of the amplified DNA fragment. Electronegative Cy5-dNTPs showed the lowest inhibitory effect on the PCR procedure, whereas electroneutral nucleotides provided a greater density of label incorporation into the growing DNA chain.