A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism
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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.
KeywordsSingle-nucleotide polymorphism Branched rolling circle amplification reaction Terpyridine–Zn(II) Fluorometry
Financial support of this work by the Ministry of Education is gratefully acknowledged.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 3.Kim S, Misra A. SNP genotyping: technologies and biomedical applications. Annu Rev Biomed Eng. 2007;9:289–320. https://doi.org/10.1146/annurev.bioeng.9.060906.152037.CrossRefGoogle Scholar
- 26.Kong F, Tong Z, Chen X, Sorrell T, Wang B, Wu Q, et al. Rapid identification and differentiation of Trichophyton species, based on sequence polymorphisms of the ribosomal internal transcribed spacer regions, by rolling-circle amplification. J Clin Microbiol. 2008;46(4):1192–9. https://doi.org/10.1128/JCM.02235-07.CrossRefGoogle Scholar
- 38.Sun Y, Lu X, Su F, Wang L, Liu C, Duan X, et al. Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer. Biosens Bioelectron. 2015;74:705–10. https://doi.org/10.1016/j.bios.2015.07.028.CrossRefGoogle Scholar