title: Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.
abstract: We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China.
authors: Dong-Shan
authors: DS
authors: Xiang
authors: Guo-Hua
authors: GH
authors: Zhou
authors: Ming
authors: M
authors: Luo
authors: Xing-Hu
authors: XH
authors: Ji
authors: Zhi-Ke
authors: ZK
authors: He
mesh_terms: D001483:Base Sequence; D052160:Benzothiazoles; D002021:Buffers; D003116:Color; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D003959:Diamines; D005240:Feasibility Studies; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D009930:Organic Chemicals / Q000737:chemistry*; D009994:Osmolar Concentration; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D052160:Benzothiazoles; D002021:Buffers; D004277:DNA, Single-Stranded; D003959:Diamines; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I