Inclusion of Molecular Beacons in PCRScorpion Primers

Molecular beacons may be used in conjunction with PCR primers to give a highly specific amplification plus detection system. Scorpion primers consist of a molecular molecular beacon A fluorescent probe molecule that contains both a fluorophore and a quenching group and that fluoresces only when it binds to a specific DNA target sequence

Scorpion primer DNA primer joined to a molecular beacon by an inert linker. When the probe sequence binds target DNA, the quencher and fluorophore are separated allowing fluorescence

FIGURE 23.23 Realtime Fluorescent PCR with TaqMan® Probe

The TaqMan® probe has three elements: a short-wavelength fluorophore on one end (diamond), a sequence that is specific for the target DNA (blue), and a long-wavelength fluorophore at the other end (circle). The two fluorophores are so close that fluorescence is quenched and no green light is emitted. This probe is designed to anneal to the center of the target DNA. When Taq polymerase elongates the second strand during PCR, its nuclease activity cuts the probe into single nucleotides. This releases the two fluorophores from contact and abolishes quenching. The short-wavelength fluorophore can now fluoresce and a signal will be detected that is proportional to the number of new strands synthesized.

Primer

Target DNA

x be

Run pcr

Probe

Probe

New DNA

New DNA

Taq polymerase

Taq polymerase

Continue elongation

Liberated green dye fluoresces

Continue elongation

Liberated green dye fluoresces

Probe DNA degraded by Taq t

Probe DNA degraded by Taq beacon (see Ch. 21) joined to a single-stranded DNA primer by an inert linker molecule (e.g. hexethylene glycol). When the beacon is in its hairpin structure, the quencher (e.g. methyl red) binds to the fluorophore (e.g. fluorescein) and prevents fluorescence. The loop portion of the stem and loop structure has sequences complementary to the target DNA, and constitutes the probe segment. When the probe sequence binds to target DNA, the hairpin is disrupted, the quencher and fluorophore are separated and fluorescence occurs.

During PCR, the Scorpion primer binds to the target DNA and is elongated by the Taq polymerase. The two strands are separated in the next denaturation cycle. The Scorpion probe sequence then hybridizes to the single-stranded DNA in the middle of the target sequence. This releases the fluorophore from the quencher and promotes fluorescence (Fig. 23.25).

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