DNA probes using fluorescence resonance energy transfer (FRET): designs and applications

Abstract

Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET The approaches used in the design of new DNA FRET probes are discussed.

Figure 1. Designs of FRET probes for hybridization detection

Adjacent hybridization probes. The fluorescence of the acceptor fluorophore is enhanced when two probes hybridize to the target sequence. Molecular beacons. Successful probe hybridization changes its configuration. The separation of the fluorophore from the quencher restores fluorescence.

Figure 2. Designs of FRET probes for PCR monitoring

Hairpin probes. They signal ongoing PCR after the hairpin is disrupted by the newly synthesized DNA strand. Scorpion primers. They change configuration and self-hybridize after the primer is extended in PCR. TaqMan probes. The probes are destroyed by Taq DNA polymerase in the course of PCR, separating the donor and acceptor fluorophores.

Figure 3

Three FRET assays for the detection of different stages in DNA recombination.

Figure 4. Deoxyribozyme biosensor

It is activated by lead ions and reports their presence by fluorescence enhancement.