Direct detection of mutant DNA in a mixed population of higher copy number wild-type DNA based on ligase detection reaction in conjunction with fluorescence resonance energy transfer

Anal Sci. 2010;26(12):1255-9. doi: 10.2116/analsci.26.1255.

Abstract

We have developed an analytical system capable of detecting point mutations in a higher copy number of wild-type DNA based on an allele-specific ligase detection reaction (LDR) in conjunction with fluorescence resonance energy transfer (FRET). Streptavidin-functionalized quantum dots (QDs) used as FRET donors effectively captured biotinylated LDR products (target DNA strands) labeled with fluorophores as a FRET acceptor, enabling the formation of a sensitive energy transfer pair and direct detection of the targets without any post-LDR separation process, which is generally required for the LDR-based mutation analysis. Our experiments indicated that the present system had an ability to detect one mutant sequence in 10 normal sequences at a signal-to-background ratio of ca. 3.9.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • DNA Mutational Analysis / methods*
  • Fluorescence Resonance Energy Transfer / methods*
  • Gene Dosage / genetics*
  • Ligases*
  • Methods
  • Point Mutation / genetics*
  • Sensitivity and Specificity

Substances

  • Ligases