A FRET-based analysis of SNPs without fluorescent probes

Nucleic Acids Res. 2004 Nov 8;32(19):e156. doi: 10.1093/nar/gnh155.

Abstract

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.

Publication types

  • Validation Study

MeSH terms

  • Benzothiazoles
  • Diamines
  • Fluorescence Resonance Energy Transfer / economics
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry
  • Gene Frequency*
  • Genotype
  • Humans
  • Organic Chemicals / chemistry
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide*
  • Quinolines
  • Sequence Analysis, DNA / economics
  • Sequence Analysis, DNA / methods*
  • Temperature

Substances

  • Benzothiazoles
  • Diamines
  • Fluorescent Dyes
  • Organic Chemicals
  • Quinolines
  • SYBR Green I