Quadratic isothermal amplification for the detection of microRNA

This protocol describes an isothermal amplification approach for ultrasensitive detection of specific microRNAs (miRNAs). It achieves this level of sensitivity through quadratic amplification of the target oligonucleotide by using a Bst DNA polymerase-induced strand-displacement reaction and a lambda exonuclease-aided recycling reaction. First, the target miRNA binds to a specifically designed molecular beacon, causing it to become a fluorescence emitter. A primer then binds to the activated beacon, and Bst polymerase initiates the synthesis of a double-stranded DNA segment templated on the molecular beacon. This causes the concomitant release of the target miRNA from the beacon - the first round of 'recycling'. Second, the duplex beacon thus produced is a suitable substrate for a nicking enzyme present in solution. After the duplex beacon is nicked, the lambda exonuclease digests the beacon and releases the DNA single strand just synthesized, which is complementary to the molecular beacon, inducing the second round of recycling. The miRNA detection limit of this protocol is 10 fmol at 37°C and 1 amol at 4°C. This approach also affords high selectivity when applied to miRNA extracted from MCF-7 and PC3 cell lines and even from breast cancer tissue samples. Upon isolation of miRNA, the detection process can be completed in ∼2 h.