Sensitive detection of active uracil-DNA glycosylase via an exonuclease Ⅲ- assisted cascade multi-amplification fluorescence DNA machine

摘要

Since uracil-DNA glycosylase (UDG) is closely related to some human diseases, monitoring and detection of UDG activity have great significance in its clinic diagnosis and functional study. Here we demonstrate a sensitive cascade multi-amplification fluorescence strategy for the active UDG assay with the help of exonuclease III (Exo III). Under the cleavage reaction of UDG, the double-stranded DNA containing peculiar uracil bases separated into two dissociative single-stranded DNA, which separately hybridized with two cyclic hairpin probes to launch the Exo Ill-assisted dual cycles. The hairpin DNA modified with fluorophore and quenching group was employed as the signal probe. After the dual cycles, countless same short DNA fragments were generated to hybridize with signal probes, initiating a new Exo Ill-assisted cyclic amplification and releasing numerous single-stranded DNA which carried only fluorophores. Thus, the fluorescence intensity of the detection system was enhanced. This study obtained the detection limit as low as 2.4 x 10(-4)U/mL for detecting the UDG activity. And it performed satisfactory selectivity and well practical applicability by analysis of the HeLa cell lysate, providing a potential method for clinic diagnosis and functional study of UDG activity.