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N. V. Komarova, S. I. Glukhov, M. S. Andrianova, A. E. Kuznetsov

Usage of fluorescent labels Cy3 and Cy5 for protection of the DNA strand against dergadation under λ exonuclease treatment

Abstract

The enzyme λ exonuclease hydrolyses 5′-phosphorylated strands of double stranded DNA in 5′ to 3′ direction. In the research λ exonuclease activity towards DNA substrates containing Cy3 and Cy5 fluorescent labels at the 5′-ends is studied. Using fluorescent method of the enzyme activity measurement it is shown that dsDNA with both 5′-ends labeled with these fluorophores doesn′t degrade under λ exonuclease treatment. The process of ssDNA production from dsDNA precursors with differently labeled 5′-ends is studied using electrophoretic separation of DNA in PAGE. Cy3 and Cy5 fluorophores at 5′-end of the DNA strand are shown to protect the strand from the enzyme cleavage both in duplex and in the single-stranded form. These labels can be used for production of fluorescently labeled single stranded DNA.
Key words: λ exonuclease, fluorescent label, preparation of single stranded DNA.
Moscow University Chemistry Bulletin.
2018, Vol. 59, No. 2, P. 92
   

Copyright (C) Chemistry Dept., Moscow State University, 2002
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