TaqMan® probe (FRET)

Recommended probe length: 15-25; Recommended length of PCR product: 50-150; G+C content: 40-60%. Avoid homo nuclear sequences and dG near the 5' end (dG acts as a weak quencher); If used for allelic discrimination, place mismatch in the centre of the probe; Ta should be 5-10 °C below probe Tm.

 

Molecular Beacons

Recommended probe length: 15-30 + arm sequences; Recommended stem length: 5-7 base pairs; Recommended amplicon length: >150; G+C content: 40-60%; Other: Avoid dG near the 5' end (dG acts as a weak quencher); Tm (beacon) approximately equal 5-7 °C above Ta (PCR). Design probe before adding flanking sequences. Adjust content and lenght of arm sequences to give Tm (stem) of about 5-7 °C above Ta. Molecular beacons will only bind to sequences that are perfectly complementary.