title: Quadratic isothermal amplification for the detection of microRNA.
abstract: 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.
authors: 1] Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan, China. [2].
authors: Ruixue
authors: R
authors: Duan
authors: 1] Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Chinese Academy of Sciences (CAS) Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, CAS, Shanghai, China. [2].
authors: Xiaolei
authors: X
authors: Zuo
authors: 1] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, CAS, Beijing, China. [2].
authors: Shutao
authors: S
authors: Wang
authors: Department of Pathology, Zhuzhou No. 1 Hospital, Hunan, China.
authors: Xiyun
authors: X
authors: Quan
authors: Department of Pathology, Zhuzhou No. 1 Hospital, Hunan, China.
authors: Dongliang
authors: D
authors: Chen
authors: Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan, China.
authors: Zhifei
authors: Z
authors: Chen
authors: Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, CAS, Beijing, China.
authors: Lei
authors: L
authors: Jiang
authors: Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility, Chinese Academy of Sciences (CAS) Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, CAS, Shanghai, China.
authors: Chunhai
authors: C
authors: Fan
authors: Key Laboratory for Large-Format Battery Materials and Systems, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan, China.
authors: Fan
authors: F
authors: Xia
mesh_terms: D020029:Base Pairing / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000302:isolation & purification*; D015335:Molecular Probes / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D035683:MicroRNAs; D015335:Molecular Probes; D004259:DNA-Directed DNA Polymerase