title: Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants.
abstract: Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as "invading stacking primer (IS-Primer)" which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Weipeng
authors.initials: W
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Minjun
authors.initials: M
authors.lastname: Zhu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: Jitao
authors.initials: J
authors.lastname: Wei
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China. Electronic address: zhouxm@scnu.edu.cn.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China. Electronic address: xingda@scnu.edu.cn.
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020641:Polymorphism, Single Nucleotide
chemical_list: D017931:DNA Primers; C524940:MIRN21 microRNA, human; D035683:MicroRNAs
keywords: Isothermal strand displacement amplification; Microrna; Single nucleotide polymorphisms; Stacking hybridization; Toehold-mediated exchange