초록 ▼

Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Method

Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Methods for quantitating amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.

대표청구항 ▼

[ We claim:] [1.] A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps ofamplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said prob

[ We claim:] [1.] A method for analyzing a target DNA sequence of a biological sample, said method comprising the steps ofamplifying the target sequence by polymerase chain reaction in the presence of two nucleic acid probes that hybridize to adjacent regions of the target sequence, one of said probes being labeled with an acceptor fluorophore and the other probe labeled with a donor fluorophore of a fluorescence energy transfer pair such that upon hybridization of the two probes with the target sequence, the donor and acceptor fluorophores are within 25 nucleotides of one another, said polymerase chain reaction comprising the steps of adding a thermostable polymerase and primers for the targeted nucleic acid sequence to the biological sample and thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature;exciting the biological sample with light at a wavelength absorbed by the donor fluorophore and detecting fluorescent emission from the fluorescence energy transfer pair.