A DNA machine for sensitive and homogeneous DNA detection via lambda exonuclease assisted amplification.

This work designs a DNA machine with three assistant DNAs and lambda exonuclease (Exo-λ) for sensitive and homogeneous fluorescent detection of DNA. The selective digestion of Exo-λ to blunt or recessed 5'-phosphorylated strand of probe 1-probe 2 duplex results in the release of target DNA and probe 2 to produce the fluorescence restoring of fluorophore labeled to probe 1. The released target DNA could hybridize with another probe 1-probe 2 duplex to trigger the target recycling for signal amplification, while the released probe 2 hybridized with molecular beacon to restore its fluorescence for signal enhancement. This DNA machine showed a fast response to target DNA with a linear concentration range from 0.4 pM to 4 nM. The limit of detection was 68 fM at a signal-to-noise ratio of 3. The high selectivity of the method may result from the Exo-λ's specific recognition-site of double-stranded DNA and the specific hybridization of target DNA with probe 1-probe 2 duplex. This DNA machine with the homogenous detection, rapid response as well as simplicity provides a new approach for sensitive detection of DNA.