Nucleic Acids Research, Vol 27, Issue 5 1316-1322, Copyright © 1999 by Oxford University Press
G Maulik, S Botchway, S Chakrabarti, S Tetradis, B Price and GM Makrigiorgos
A highly sensitive method to detect traces of aldehyde-containing
apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based
on this method, a novel approach to detect DNA base mismatches recognized
by the mismatch repair glycosylase MutY is demonstrated. Open chain
aldehydes generated in nucleic acids due to spontaneous depurination, DNA
damage or base excision of mismatched adenine by MutY are covalently
trapped by a new linker molecule [fluorescent aldehyde- reactive probe
(FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing
AP sites is FARP-trapped, biotinylated and immobilized onto
neutravidin-coated microplates. The number of FARP- trapped aldehydes is
then determined via chemiluminescence using a cooled ICCD camera. AP sites
induced in plasmid or genomic calf thymus DNA via mild depurination or by
simple incubation at physiological conditions (pH 7, 37 degreesC) presented
a linear increase in chemiluminescence signal with time. The procedure
developed, from a starting DNA material of approximately 100 ng, allows
detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7)
bases, and extends by 1-2 orders of magnitude the current limit in AP site
detection. In order to detect MutY-recognized mismatches, nucleic acids are
first treated with 5 mM hydroxylamine to remove traces of spontaneous
aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides
engineered to have a centrally located A/G mismatch demonstrate a strong
chemiluminescence signal. Similarly, single- stranded M13 DNA that forms
mismatches via self-complementation (average of 3 mismatches over 7429
bases) and treated with MutY yields a signal approximately 100-fold above
background. No signal was detected when DNA without mismatches was used.
The current development allows sensitive, non-isotopic, high throughput
screening of diverse nucleic acids for AP sites and mismatches in a
microplate-based format.
ARTICLES
Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes
Dana Farber Cancer Institute, Joint Center for Radiation Therapy, Department of Radiation Oncology,Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.
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