Palindromic molecular beacon-based intramolecular strand-displacement amplification strategy for ultrasensitive detection of K-ras gene

Abstract The sensitive detection of tumor proto-oncogenes is indispensable because the early diagnosis and accurate treatment of genetic diseases is the key guarantee of patients' health. In this study, we proposed a novel palindromic molecular beacon (PMB) that it bases on the signal amplification strategy for ultrasensitive detection of Kras gene codon 12. PMB is designed to have two palindromic fragments at its two ends, one of which is locked via folding into a hairpin structure and the other promotes the formation of PMB duplex via intermolecular self-hybridization. Target DNA can hybridize to the loop portion of PMB and release the palindromic fragment at the 3’ end. Within the PMB duplex, the two palindromic fragments released hybridize with each other and serve as polymerization primer responsible for the strand-displacement amplification (SDA). Namely, hybridized target DNA can be displaced and initiates the next round of reactions, making the polymerization/displacement/hybridization process go forward circularly. As a result, a large number of polymerization products are produced, dramatically enhancing optical signal. Because primer hybridization and polymerization-based displacement occur within PMB duplex, the reaction process is called intramolecular strand-displacement amplification (ISDA). Via utilizing the newly-proposed PMB-based ISDA strategy, the target K-ras gene could be detected down to 10 pM with a wide response range of 1 × 10 −11 -1.5 × 10 −7  M, and point mutations are easily distinguished, realizing the ultrasensitive, highly selective detection of K-ras gene. This impressive sensing paradigm demonstrates a new concept of signal amplification for the detection of disease-related genes only via using a simple way to efficiently amplify optical signal.