Looking for a simple, accurate nuclease assay

Frank Moersberger frank.moersberger at merck.de
Wed Sep 8 03:27:43 EST 1999


I'm looking for a simple, sensitive and accurate endonuclease
(unspecific) assay. The assay we use so far, is based on sonicated
salmon sperm DNA. After incubation the nuclease reaction is stopped by
TCA precipitation. After centrifugation the acid-soluble oligos are then
measured photometrically at 260nm.
This assay worked well
However, we want  to overcome the inter-laboratory variations (We assume
the precipitation is critical) and also want to improve the accuracy of
the nuclease assay (c.v = 10%).

The BEACON DNase assay offered by PanVera  is much too sensitive for our
purposes, although the principle would be ideal. A circular DNA labeled
with a fluorescence tag and a quencher group in close neighbourhood is
used as substrate. Upon nuclease action the ring is relaxed separating
dye and quencher from each other such causing a strong fluorescence
signal.
Do you have any idea whether a less sensitive, but also comparable
photometric tests is on the market? Or do you have any other idea, how
we might improve our assay?
Thanks
F.M..




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