A dual-signal amplification method for the DNA detection based on exonuclease III

A dual-signal amplification method based on two molecular beacons was designed for human hemochromatosis (HFE) gene detection. The two probes, P1 and P2, could resist the exonuclease III (Exo III) digestion due to the 3'-termini protrusion, and could coexist stably with Exo III. In the presence of HFE targets, P1 hybridized with a HFE target to form a duplex DNA with a recessed 3'-hydroxyl termini and then partially digested by Exo III, releasing the HFE target and a residual sequence (X). This X sequence could trigger the digestion of P2 probes with 6-carboxy-fluoresceins and Black Hole Quenchers and then result in the increase of fluorescence intensity. The X sequences were more stable than HFE targets and could cyclically trigger the P2 digestion for a long time even though the HFE targets were digested by Exo III. This method improved the sensitivity and reached 4 orders of magnitude in detection limit, and showed excellent selectivity to discriminate single base mismatched targets well.