Monitoring a fluorescence energy transfer pair during hybridization of first probe labelled with fluorescein to second probe labelled with Cy5 or Cy5.5

摘要

Methods of monitoring hybridisation during polymerase chain reaction are described. These methods are achieved with rapid thermal cycling and the use of double stranded DNA dyes or specific hybridisation probes. A fluorescence resonance energy transfer pair comprising a first probe labeled with fluorescein and a second probe labeled with Cy5 or Cy5.5. A fluorescence resonance energy transfer comprising a donor fluorophore having an emission spectrum and an acceptor fluorophore having an absorption spectrum and extinction coefficient greater than 100,000 M-1cm-1, wherein the donor fluorophore's emission spectrum and the acceptor fluorophore absorption spectrum overlap less than 25%. Methods for quantifying amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.