The G-BHQ synergistic effect: Improved double quenching molecular beacons based on guanine and Black Hole Quencher for sensitive simultaneous detection of two DNAs

Publication date: 1 November 2017
Source:Talanta, Volume 174
Author(s): Dongshan Xiang, Fengquan Li, Chenyi Wu, Boan Shi, Kun Zhai
We designed two double quenching molecular beacons (MBs) with simple structure based on guanine (G base) and Black Hole Quencher (BHQ), and developed a new analytical method for sensitive simultaneous detection of two DNAs by synchronous fluorescence analysis. In this analytical method, carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were respectively selected as fluorophore of two MBs, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were respectively selected as organic quencher, and three continuous nucleotides with G base were connected to organic quencher (BHQ-1 and BHQ-2). In the presence of target DNAs, the two MBs hybridize with the corresponding target DNAs, the fluorophores are separated from organic quenchers and G bases, leading to recovery of fluorescence of FAM and TAMRA. Under a certain conditions, the fluorescence intensities of FAM and TAMRA all exhibited good linear dependence on their concentration of target DNAs (T1 and T2) in the range from 4 × 10⁻¹⁰ to 4 × 10⁻⁸molL⁻¹ (M). The detection limit (3σ, n = 13) of T1 was 3 × 10⁻¹⁰M and that of T2 was 2×10⁻¹⁰M, respectively. Compared with the existing analysis methods for multiplex DNA with MBs, this proposed method based on double quenching MBs is not only low fluorescence background, short analytical time and low detection cost, but also easy synthesis and good stability of MB probes.

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