Discrimination of the false-positive signals of molecular beacons by combination of heat inactivation and using single walled carbon nanotubes

Abstract

Molecular beacons (MBs) have been extensively used for real-time monitoring of RNA/DNA and protein molecules. However, such versatility also brings about multiple sources of positive signals. Moreover, the covalently attached quencher or fluorophore may even be cleaved from the strand by the exonucleases, followed by complete degradation of the probe. These undesirable false-positive signals (FPSs) have seriously limited the application of MBs to detect real world samples. In this paper, we propose a novel and efficient approach for discrimination of FPSs of MBs due to non-specific MB-protein interactions and nuclease degradation by combination of heat inactivation and using single walled carbon nanotubes (SWNTs). The mechanisms of different DNA-protein interactions that are responsible for the generation of FPSs of MBs were investigated in detail. The proposed strategy can quickly identify the possible sources of FPSs caused by mechanisms other than hybridization in detecting real samples, which would be very helpful in choosing a proper way to modify the structure of the MBs or using a specific inhibitor. The established method was successfully applied to verify the FPSs in the measurement of a plant tissue sample.