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

Kenjiro Kosaki, Hiroshi Yoshihashi, Yuko Ohashi, Rika Kosaki, Taichi Suzuki, Nobutake Matsuo

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)111-119
Number of pages9
JournalJournal of Biochemical and Biophysical Methods
Volume47
Issue number1-2
DOIs
Publication statusPublished - 2001 Jan 30

Keywords

  • DHPLC
  • Imprinting
  • Methylation
  • SNuPE

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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