Thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection

Detection of point mutations in driver genes is of great significance for the early diagnosis, treatment, and prognostic evaluation of cancer. However, current detection methods do not offer versatility, specificity, and rapid performance simultaneously. Thus, multiple mutation detection processes are necessary, which results in long processing times and high costs. In this study, we developed a thermodynamics-guided two-way interlocking DNA cascade system for universal multiplexed mutation detection (TTI-CS). This strategy is based on the DNA probe, which changes the thermodynamic balance of the DNA cascade by the designed bubble structure, thereby achieving a good distinction between mutant and wild-type DNA. The designed method greatly shortens the detection time through two-way intrusion. In addition, this method only changes two inexpensive trigger and bridge sequences, which replace the specific and expensive nucleic acid probes used in analyses based on traditional DNA probe methods, thereby enabling multiple detections. We performed the detection of synthetic single-stranded DNA for the five mutation points and successfully detected in endometrial cancer specimens. The detection limit of this method is 0.1%, which better meets the needs of clinical low-abundance multiple mutation detection. Overall, TTI-CS is currently one of the best methods for detecting multiple mutation detections.