Nucleic Acids Research, Vol 26, Issue 6 1515-1521, Copyright © 1998 by Oxford University Press
AV Fotin, AL Drobyshev, DY Proudnikov, AN Perov and AD Mirzabekov
A microchip method has been developed for massive and parallel
thermodynamic analyses of DNA duplexes. Fluorescently labeled
oligonucleotides were hybridized with oligonucleotides immobilized in the
100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting
curves for all microchip duplexes were measured in real time in parallel
for all microchip duplexes. Thermodynamic data for perfect and mismatched
duplexes that were obtained using the microchip method directly correlated
with data obtained in solution. Fluorescent labels or longer linkers
between the gel and the oligonucleotides appeared to have no significant
effect on duplex stability. Extending the immobilized oligonucleotides with
a four-base mixture from the 3'-end or one or two universal bases
(5-nitroindole) from the 3'- and/or 5'- end increased the stabilities of
their duplexes. These extensions were applied to increase the stabilities
of the duplexes formed with short oligonucleotides in microchips, to
significantly lessen the differences in melting curves of the AT- and
GC-rich duplexes, and to improve discrimination of perfect duplexes from
those containing poorly recognized terminal mismatches. This study explored
a way to increase the efficiency of sequencing by hybridization on
oligonucleotide microchips.
ARTICLES
Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips
Joint Human Genome Program: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 117984 Moscow, Russia.
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