Typing and determination of SNP functional gene based on highly selective and signal-amplified fluorescence double-probe with the help of ExoIII nuclease and magnetic bead

• Published in: Journal of pharmaceutical and biomedical analysis 2020-02, Vol.179, p.112917-112917, Article 112917
• Main Authors: Min, Chun-Yan, Wu, Lu-Qian, Qian, Ting-ting, Ul Ain, Noor, Liu, Wen-Juan, Wu, Xiao-Ning, Zhang, Chen, Chen, Zhe, Xie, Hong-Ping
• Format: Article
• Language: English
• Subjects: ExoIII nuclease; Fluorescence double-probe; Gene determination; Genotyping; Magnetic bead; SNP
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2019.112917

[Display omitted] •A highly selective signal-amplified fluorescence double-probe detection system was constructed.•Three fluorescence phenotypes with significant difference were produced for accurate SNP genotyping.•Fluorescence signal was amplified up to 6063 times for sensitive determination of wild- and mutant-type genes.•Same probe could be used in both qualitative genotyping and quantitative gene determination for SNP gene. We have developed a fluorescence double-probe detection system with signal amplification for simple typing and determination of single nucleotide polymorphism (SNP) functional gene based on non-sequence dependence of ExoIII nuclease on dsDNA and rapid separation of magnetic bead. Matched detected gene can cyclically release abundant fluorescence-labeled ssDNA from the probe and the corresponding measured fluorescence signal is amplified up to 6063 times. In this case, the probe cannot release the measured fluorescence signal for the point mutation gene and then the corresponding measured signal is inhibited. According to signal amplification and inhabitation of the probe, we proposed both an accurate genotyping approach with strong specificity and a sensitive determination approach with high selectivity for SNP functional gene. For qualitative genotyping, there are obvious genotype-based differences of measured fluorescence phenotypes among three kinds of the samples of the investigated SNP. The quantitative determinations of its wild-type gene and mutant gene have all a good linearity in the range from 0.5 to 500 pmol/L with the correlation coefficients R2 of 0.9940 and 0.9911, and a high sensitivity with the detection limits of 0.11 and 0.20 pmol/L, respectively. Compared to the usual single-probe detection system, the developed double-probe system can achieve not only accurate genotyping but also the sensitive gene determination. Meanwhile, it is also a simple and reliable method for both quantitative and qualitative analysis of functional gene.