Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon

Yang-Wei Lin, Hsin Tsung Ho, Chih Ching Huang, Huan Tsung Chang

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

We present a simple and novel assay - employing a universal molecular beacon (MB) in the presence of Hg2+ - for the detection of single nucleotide polymorphisms (SNPs) based on Hg2+-DNA complexes inducing a conformational change in the MB. The MB (T7-MB) contains a 19-mer loop and a stem of a pair of seven thymidine (T) bases, a carboxyfluorescein (FAM) unit at the 5′-end, and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) unit at the 3′-end. Upon formation of Hg2+ -T7-MB complexes through T-Hg2+-T bonding, the conformation of T7-MB changes from a random coil to a folded structure, leading to a decreased distance between the FAM and DABCYL units and, hence, increased efficiency of fluorescence resonance energy transfer (FRET) between the FAM and DABCYL units, resulting in decreased fluorescence intensity of the MB. In the presence of complementary DNA, double-stranded DNA complexes form (instead of the Hg2+ -T7-MB complexes), with FRET between the FAM and DABCYL units occurring to a lesser extent than in the folded structure. Under the optimal conditions (20 nM T7-MB, 20 mM NaCl, 1.0 μM Hg2+, 5.0 mM phosphate buffer solution, pH 7.4), the linear plot of the fluorescence intensity against the concentration of perfectly matched DNA was linear over the range 2-30 nM (R2 = 0.991), with a limit of detection of 0.5 nM at a signal-to-noise ratio of 3. This new probe provides higher selectivity toward DNA than that exhibited by conventional MBs.

Original languageEnglish
Article numbere123
JournalNucleic Acids Research
Volume36
Issue number19
DOIs
Publication statusPublished - 2008 Nov 19

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Single Nucleotide Polymorphism
Fluorescence
Fluorescence Resonance Energy Transfer
DNA
Molecular Conformation
Benzoic Acid
Signal-To-Noise Ratio
Thymidine
Limit of Detection
Buffers
Complementary DNA
Phosphates
4-(4-dimethylaminophenylazo)benzoic acid

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Lin, Yang-Wei ; Ho, Hsin Tsung ; Huang, Chih Ching ; Chang, Huan Tsung. / Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon. In: Nucleic Acids Research. 2008 ; Vol. 36, No. 19.
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Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon. / Lin, Yang-Wei; Ho, Hsin Tsung; Huang, Chih Ching; Chang, Huan Tsung.

In: Nucleic Acids Research, Vol. 36, No. 19, e123, 19.11.2008.

Research output: Contribution to journalArticle

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