Use of the Cy3 and Cy5 Fluorescent Labels to Protect a DNA Strand from Degradation under λ Exonuclease Treatment

Resumo

λ Exonuclease hydrolyzes a 5′-phosphorylated strand of double-stranded DNA in the 5′–3′ direction. In this paper, the activity of the enzyme with respect to DNA substrates containing Cy3 and Cy5 fluorescent labels at their 5′ ends is studied for the first time. It is demonstrated using the fluorescence procedure for measuring the exonuclease activity that double-stranded DNA, of which both the 5′ ends contain these fluorophores, is not destroyed under the action of λ exonuclease. Using the electrophoretic separation of DNA in polyacrylamide gel, the process of obtaining single-stranded DNA from double-stranded precursors containing different labels at the 5′ ends is studied. It is shown that the introduction of Cy3 and Cy5 fluorophores to the 5′ end of the DNA strand protects this strand from enzyme damage both in the duplex and in single-stranded form, and these labels can be used to produce fluorescently labeled single-stranded DNA.