Improving the sensitivity and selectivity of a DNA probe using graphene oxide-protected and T7 exonuclease-assisted signal amplification

The accurate analysis of single-nucleotide polymorphisms is of great significance for clinical detection and diagnosis. Based on the hybridization hindrance caused by graphene oxide (GO) and hairpin probe, we report a T7 Exo-assisted cyclic amplification technique to distinguish single-base mismatch for highly sensitive and selective detection of mutant-type DNA. When the mutant-type target is completely complementary to the probe, the T7 Exo hydrolyzes the probe and releases the fluorescent molecule from the GO surface, resulting in a fluorescence signal. Conversely, when the wild-type mismatch target is present, the weak hybridization prevents the release of FAM-labeled probe from the GO surface. Therefore, the FAM-labeled probe cannot be degraded efficiently by T7 Exo, and the fluorescence is still quenched by GO. The detection limit of the proposed method can be as low as 34 fM due to the cyclic signal amplification. The experimental results showed that the established method could be used to detect single-nucleotide polymorphisms accurately and sensitively at low cost.