Real-time Detection of Specific Fragments of DNA or RNA with the Help of FRET-based Polymerase,

Ligase and Hybridization Chain Reactions

Chemeris A.V., Nikonorov Yu.M., Chemeris D.A., Romanenkova M.L.,

Matniyazov R.T., Knyazev A.V., Gimalov F.R., Vakhitov V.A.

Institute of Biochemistry and Genetics USC RAS, Ufa, Russia

 

We have elaborated a better method of real-time PCR on the platform "UFA" (Universal Fluorescent Amplification), where connivent annealing of fluorescent labeled primers results in FRET. In such case besides other advantages it is possible to amplify short fragments of DNA or RNA which with the greater probability can be kept in samples old or undergone to a strong destructive influence. Similar approach with quenching of the fluorescence was suggested for thermocyclers where no FRET registration. We have elaborated a method of amplicon detection in real-time LCR, based on FRET between a dye-donor and a dye-acceptor attached to adjacent oligonucleotides which during LCR become a joint molecule. HCR feature is that there is no amplification DNA or RNA fragments, but takes place an isothermal self-assembly of nanoconstructions carrying FRET donor-acceptor pair of dyes, initiated only completely complementary sequences and testifying about presence of those. HCR driving force is temporarily latent energy, made in special designed oligonucleotides.