title: Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.
abstract: Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples.
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
authors.forename: Yongshun
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
authors.forename: Xingti
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China. Electronic address: wangl-sdu@sdu.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: wjiang@sdu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
mesh_terms: D020137:Base Pair Mismatch; D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000032:analysis / Q000235:genetics*; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D013050:Spectrometry, Fluorescence / Q000379:methods*; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Fluorescent detection; Isothermal DNA amplification; Single-base mutation; Toehold-mediated strand displacement reaction