We have investigated the possibility of determining quantitatively the alle
les of binary DNA polymorphisms by single-nucleotide primer extension (SNuP
E) and fluorescence-based DHPLC. Using a polymorphism of interest to our gr
oup, ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of
the primer annealed to the template adjacent to the polymorphic site. The p
rimer extension product was then resolved from the unincorporated dye termi
nator by ion-pair reversed-phase liquid chromatography. The signal intensit
y of incorporated ROX-ddCTP correlated well over one order of magnitude wit
h the relative amount of the C-allele present in the genomic DNA template.
We conclude that SNuPE, when combined with fluorescence-based DHPLC, can ac
curately determine the relative molar proportion of one allele in total DNA
. (C) 2001 Elsevier Science B.V. All rights reserved.