Loopback rolling circle amplification for ultrasensitive detection of Kras gene

Abstract Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100 fM to 20 nM), and the detection limit is down to 16 fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics.