Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension

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.