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Molecular Beacons In Multiplex Digital PCR Assays

PCR, molecular beacon, molecular probe, Kramer

Every droplet of a digital PCR assay can contain as many as 35 different highly specific molecular beacon probes that are each tagged with a different combination of three out of seven available fluorescent colours. All the molecular beacons remain dark except for those whose probe sequence is exactly complementary to a target sequence within the amplicons that are generated in a droplet. Because each droplet is a screening assay, as many as 35 rare mutations can be simultaneously and accurately quantitated with an instrument distinguishing the fluorescent colours that are present.

At the 2nd qPCR and Digital PCR Congress: USA, Fred Kramer explained how to detect the 35 different possible kinds of amplicons that can be developed in digital PCR via the colour-coded molecular beacons developed in his lab.

Structure of colour-coded molecular beacons

These molecular beacons show a fluorophore located next to a quencher of fluorescence, making them dark and therefore not emitting any signals. However, if a target is present (e.g. an amplicon made in a PCR reaction whose sequence is complimentary to the sequence on the probe) the molecular beacon will bind to form a hybrid with the amplicon. This reaction separates the quencher fluorescence from the fluorophore allowing the fluorophore to emit bright signals. The molecular beacon is a single-stranded oligonucleotide forming a hairpin structure. The loop of its hairpin is the sequence complementary to the target amplicon. It’s embedded within two arms which are unrelated to the target in the amplicon. These are complimentary to each other, hence when no target is found around, it forms a hairpin and covalently link to one end of the oligonucleotide via a flexible linker.

On the other end of the oligonucleotide exists a quencher of fluorescence and if the quencher is close enough to the fluorophore, the energy that is absorbed by the fluorophore is transferred to the quencher, making all the structure dark and the size of the fluorophore and the size of the quencher result like the one of the individual nucleotide. The operation principle of a molecular beacon is to bind to the target sequence to form a probe target hybrid.

Applications of molecular probes

The designed probe, due to the rigidity of the nucleic acid double helices, could separate the arms of the fluorophore and quencher leading to a bright fluorescence. Hence, the classic use of the molecular beacon is to be added to the PCR reaction prior to the start of the reaction. As the amplicons are made, the molecular beacons bind to those amplicons and fluoresce brightly and through the intensity of fluorescence it is possible to increase the amplicon of interest.

Finally, molecular beacons are extraordinarily specific and sensitive as they present two possible stable states; one is to act as a hairpin and the other is to bind to the target making it a stable hybrid. Molecular beacons of this type can facilitate the detection of specific sequences of nucleic acids and therefore targeting different rare mutations associated with cancer, diagnosis and other treatments.

qPCR, PCR, molecular beaker
Fred Kramer is a Professor of Microbiology, Biochemistry and Molecular Genetics at Rutgers University. He will speak at the 3rd qPCR and Digital PCR Congress: USA.

 

Hear more about other applications of PCR in oncology, clinical applications and infectious diseases. Join the qPCR and Digital PCR strand of the 4BIO Summit.

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