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A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism

Ma, Qian, Gao, Zhiqiang
Analytical and bioanalytical chemistry 2018 v.410 no.13 pp. 3093-3100
DNA, fluorescence, gene amplification, genes, mutants, nucleotides, single nucleotide polymorphism
In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine–Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine–Zn(II)–pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstract A simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine–Zn(II) complex.