title: Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.
abstract: A highly sensitive fluorescence method of quantitative detection for speciﬁc DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.
authors.affiliation: Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei Minzu University, Enshi 445000, China; School of Chemical and Environmental Engineering, Hubei Minzu University, Enshi 445000, China.
authors.forename: Dongshan
authors.initials: D
authors.lastname: Xiang
authors.affiliation: School of Chemical and Environmental Engineering, Hubei Minzu University, Enshi 445000, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhai
authors.affiliation: Sichuan University of Arts and Science, Dazhou, 635000, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Xiang
authors.affiliation: School of Chemical and Environmental Engineering, Hubei Minzu University, Enshi 445000, China.
authors.forename: Lianzhi
authors.initials: L
authors.lastname: Wang
mesh_terms: D000476:Alkanesulfonates / Q000737:chemistry*; D001391:Azo Compounds / Q000737:chemistry*; D052160:Benzothiazoles; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D003959:Diamines; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008027:Light; D057230:Limit of Detection; D009696:Nucleic Acids / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry*; D011804:Quinolines; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000476:Alkanesulfonates; D001391:Azo Compounds; C000598590:BHQ-1 compound; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; C024098:6-carboxyfluorescein; D004247:DNA
keywords: Highly sensitive fluorescence detection; Molecular beacons; SYBR Green I; Sequence-speciﬁc DNA