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