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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.

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Figure 1: Structure of the self-hybridized molecular beacon probe 21, in which are reported the matching nucleotides of miR-21 and probe 21's primer.
Figure 2: Overview of the HQEA strategy.
Figure 3: Results from various experiments with different reaction components.
Figure 4: Sensitivity of the HQEA approach at different reaction temperatures.
Figure 5: Sensitivity performance of HQEA in the detection of miRNA extracts from cancer cell lines.
Figure 6: Specificity of probes 21 and 221 for the detection of target miRNAs with or without mismatching bases.
Figure 7: Specificity of probe 21 and probe 221 for different cell types.
Figure 8: Selectivity and accuracy of the HQEA approach for the detection of miRNA extracted from human tissues.

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This research was supported by initiatory financial support from HUST, the National Basic Research Program of China (2013CB933000), the 100 Talents Program from the CAS, the Shanghai Pujiang Project (grant no. 13PJ1410700 to X. Z.), the 1,000 Young Talents program (to F. X.) and the National Natural Science Foundation (21375042).

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R.D., F.X., X.Z., L.J., S.W., Z.C., C.F., X.Q. and D.C. conceived the projects, designed and conducted experiments, analyzed the data and wrote the manuscript. R.D., X.Z. and S.W. contributed equally to this work.

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Correspondence to Fan Xia.

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The authors declare no competing financial interests.

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Duan, R., Zuo, X., Wang, S. et al. Quadratic isothermal amplification for the detection of microRNA. Nat Protoc 9, 597–607 (2014).

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