Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

Charles R. Twist, Michael Kenneth Winson, Jeremy John Rowland, Douglas B. Kell

Research output: Contribution to journalArticlepeer-review

21 Citations (SciVal)

Abstract

We have exploited three methods for discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation or otherwise of labeled dideoxy nucleotides at the end of a primer chain using single-molecule fluorescence detection methods. Good discrimination of incorporated vs free nucleotide may be obtained in a homogeneous assay (without washing steps) via confocal fluorescence correlation spectroscopy or by polarization anisotropy obtained from confocal fluorescence intensity distribution analysis. Moreover, the ratio of the fluorescence intensities on each polarization channel may be used directly to discriminate the nucleotides incorporated. Each measurement took just a few seconds and was done in microliter volumes with nanomolar concentrations of labeled nucleotides. Since the confocal volumes interrogated are 1 fL and the reaction volume could easily be lowered to nanoliters, the possibility of SNP analysis with attomoles of reagents opens up a route to very rapid and inexpensive SNP detection. The method was applied with success to the detections of SNPs that are known to occur in the BRCA1 and CFTR genes.
Original languageEnglish
Pages (from-to)35-44
Number of pages10
JournalAnalytical Biochemistry
Volume327
Issue number1
DOIs
Publication statusPublished - 01 Apr 2004

Keywords

  • SNP
  • Detection
  • FCS
  • Single molecule
  • Miniaturization
  • Assay

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