title: Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension.
abstract: We have investigated the possibility of determining quantitatively the alleles of binary DNA polymorphisms by single-nucleotide primer extension (SNuPE) and fluorescence-based DHPLC. Using a polymorphism of interest to our group, 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 primer extension product was then resolved from the unincorporated dye terminator by ion-pair reversed-phase liquid chromatography. The signal intensity of incorporated ROX-ddCTP correlated well over one order of magnitude with the relative amount of the C-allele present in the genomic DNA template. We conclude that SNuPE, when combined with fluorescence-based DHPLC, can accurately determine the relative molar proportion of one allele in total DNA.
authors.affiliation: Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan. kkoskai@med.keio.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Kosaki
authors.forename: H
authors.initials: H
authors.lastname: Yoshihashi
authors.forename: Y
authors.initials: Y
authors.lastname: Ohashi
authors.forename: R
authors.initials: R
authors.lastname: Kosaki
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: N
authors.initials: N
authors.lastname: Matsuo
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes; D051383:GRB10 Adaptor Protein; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011506:Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D011506:Proteins; D051383:GRB10 Adaptor Protein; D004247:DNA