Shared-probe system: An accurate, low-cost and general enzyme-assisted DNA probe system for detection of genetic mutation

ABSTRACT Enzyme assisted DNA probes are powerful tools in molecular diagnostics for their simplicity, rapidity, and low detection limit. However, cost of probes, difficulty in optimization and disturbance of secondary structure hindered the wider application of enzyme assisted DNA probes. To solve the problems, we designed a new system named shared-probe system. By introducing two unlabeled single stranded DNA named Sh1 and Sh2 as the bridge between probe and the substrate, the same sequence of dually labeled probe with stable performance was shared for different mutations, thus sparing the expense and time cost on designing, synthesizing and optimizing corresponding probes. Besides, the hybridization between Sh1 and the substrate could overcome secondary structures, which guaranteed the detection of different substrates. The performance and generality of the design were tested by low abundance detection in synthetic single DNA samples and the limit of detection was 0.05% for PTENR130Q, EGFR-L858R and 0.02% for BRCA1-NM007294.3. In genomic DNA samples, the limit of detection of 0.1% can be achieved for EGFR-L858R, demonstrating the potential of clinical application in our design.