Molecular Beacon
Molecular beacons are special hybridization probes used in molecular biology to identify and quantify DNA . They consist of a single-stranded DNA with a stem-loop or hairpin structure , at the 3 'and 5' ends of which belong to the stem a fluorescent dye or a quencher is located. The loop, however, contains a DNA sequence complementary to the target DNA . A hybridization with the target DNA causes an increase in fluorescence, the analytic example in Hybridisationsanalysen and in the quantitative real-time PCR is used.
structure
Molecular beacons usually consist of 25 to 40 nucleotides and have a stem-loop structure. The 3 'and 5' ends, which are complementary to one another and consist of 5 to 8 base pairs, form the stem. The loop consists of a 15 to 35 base pair long nucleic acid sequence complementary to the target DNA. The 3 'and 5' ends are coupled with a fluorescent dye or a quencher, usually Dabcyl . The use of a second fluorescent dye instead of the quencher is possible but not common.
function
In the unhybridized state at room temperature, molecular beacons are in their stem-loop structure. The fluorescence of the fluorescent dye (donor) is suppressed by an energy transfer based on the Förster resonance energy transfer to the quencher (acceptor). A hybridization of molecular beacons with the target DNA causes a dissolution of their tertiary structures . An increase in donor fluorescence after excitation can be observed through a decrease in energy transfer as a result of hybridization of the molecular beacons with the target DNA.
literature
- Tyagi S, Kramer FR: Molecular beacons: probes that fluoresce upon hybridization . In: Nat. Biotechnol. . 14, No. 3, March 1996, pp. 303-8. doi : 10.1038 / nbt0396-303 . PMID 9630890 .