Citation: LU Zi-Jing, XIONG Wei-Wei, ZHAI Kun, XIANG Dong-Shan, TAN Zhi-Dou. Dual Color Fluorescence Quantitative Detection of DNA Sequences with Double-quenching Molecular Beacons and Nucleic Acid Hoechst 33258[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(7): 1014-1020. doi: 10.19756/j.issn.0253-3820.181791 shu

Dual Color Fluorescence Quantitative Detection of DNA Sequences with Double-quenching Molecular Beacons and Nucleic Acid Hoechst 33258

LU Zi-Jing ^1,2, ,
XIONG Wei-Wei ^1,2 ,
ZHAI Kun ^1,2 ,
XIANG Dong-Shan ¹ ,
TAN Zhi-Dou ²

1.
Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities, Enshi 445000, China;
2.
School of Chemical and Environmental Engineering, Hubei University for Nationalities, Enshi 445000, China

Received Date: 20 December 2018
Revised Date: 6 May 2019

Fund Project: This work was supported by the National Natural Science Foundation of China (No. 21465010).

A double-quenching molecular beacons (MB) was designed based on dual quenching of organic quencher Black Hole Quencher 1 (BHQ-1) and guanine (G) bases to fluorophore 6-carboxyfluorescein group (FAM). A dual color fluorescence quantitative detection method for specific single-stranded DNA sequences has been developed based on MB and nucleic acid dye hoechst 33258. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In this MB, FAM and BHQ-1 are respectively selected as fluorophore and organic quencher, three continuous nucleotides with G base are connected with BHQ-1, the bases of the stem in MB is completely designed as C-G base pairs, and the base sequence of the loop in MB is designed as the complementary sequence of target DNA. In the absence of target DNA, the MBs is the stem-loop structure, the FAM is close to BHQ-1 and G bases, the fluorescence of FAM is dually quenched by BHQ-1 and G bases, and the fluorescence of FAM is very weak. In addition, the C-G base pairs of MB cannot be combined with Hoechst 33258, so the fluorescence of Hoechst 33258 is also very weak. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strand structure, the stem-loop structure of MB is destroyed, and the FAM is separated with BHQ-1 and G bases, leading to recovery of fluorescence of FAM. At the same time, the nucleic acid dye Hoechst 33258 binds to the A-T base pair in double-stranded DNA, and its fluorescent signal is significantly enhanced. Thus, a dual color fluorescence quantitative detection for the target DNA can be realized through the fluorescence enhancement of FAM and Hoechst 33258. Under optimized conditions, the total fluorescence intensities of FAM and Hoechst 33258 exhibit a good linear dependence on concentration of target DNA in the range of 0.05-8.0 nmol/L, and the regression equation is ΔI_T=192.2C + 115.08 (R²=0.9938) with detection limit of 20 pmol/L (3σ, n=9). The method has simple operation, high sensitivity, good selectivity and low detection limit.
- Double-quenching molecular beacon,
- Single stranded DNA,
- Hoechst 33258,
- Dual color fluorescence,
- Quantitative detection
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