Nucleic Acids Research Advance Access originally published online on August 27, 2008
Nucleic Acids Research 2008 36(19):e123; doi:10.1093/nar/gkn537
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Nucleic Acids Research, 2008, Vol. 36, No. 19 e123
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Fluorescence detection of single nucleotide polymorphisms using a universal molecular beacon
1Department of Chemistry, National Taiwan University 1, Section 4, Roosevelt Road, 2Department of Laboratory Medicine, Mackay Memorial Hospital, 3Mackay Medicine, Nursing and Management College, Taipei and 4Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan
*To whom correspondence should be addressed. Tel: +886 2 33661171; Fax: +886 2 33661171; Email: changht{at}ntu.edu.tw
Received March 27, 2008. Revised August 5, 2008. Accepted August 6, 2008.
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.