Lin Yang-Wei, Ho Hsin-Tsung, Huang Chih-Ching, Chang Huan-Tsung
Department of Chemistry, National Taiwan University 1, Taipei, Taiwan.
Nucleic Acids Res. 2008 Nov;36(19):e123. doi: 10.1093/nar/gkn537. Epub 2008 Aug 27.
We present a simple and novel assay-employing a universal molecular beacon (MB) in the presence of Hg(2+)-for the detection of single nucleotide polymorphisms (SNPs) based on Hg(2+)-DNA complexes inducing a conformational change in the MB. The MB (T(7)-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 Hg(2+)-T(7)-MB complexes through T-Hg(2+)-T bonding, the conformation of T(7)-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 Hg(2+)-T(7)-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 T(7)-MB, 20 mM NaCl, 1.0 muM Hg(2+), 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 (R(2) = 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.
我们展示了一种简单且新颖的检测方法——在汞离子（Hg(2+)）存在的情况下使用通用分子信标（MB）——用于基于Hg(2+) - DNA复合物诱导分子信标构象变化来检测单核苷酸多态性（SNP）。该分子信标（T(7)-MB）包含一个19聚体环和一对七个胸腺嘧啶（T）碱基的茎，5'端有一个羧基荧光素（FAM）单元，3'端有一个4 - （[4 - （二甲基氨基）苯基]偶氮）苯甲酸（DABCYL）单元。通过T - Hg(2+) - T键形成Hg(2+) - T(7)-MB复合物后，T(7)-MB的构象从无规卷曲变为折叠结构，导致FAM和DABCYL单元之间的距离减小，从而提高了FAM和DABCYL单元之间荧光共振能量转移（FRET）的效率，导致分子信标荧光强度降低。在互补DNA存在的情况下，会形成双链DNA复合物（而不是Hg(2+) - T(7)-MB复合物），FAM和DABCYL单元之间的FRET程度比在折叠结构中要小。在最佳条件（20 nM T(7)-MB、20 mM NaCl、1.0 μM Hg(2+)、5.0 mM磷酸盐缓冲溶液，pH 7.4）下，荧光强度相对于完全匹配DNA浓度的线性图在2 - 30 nM范围内呈线性（R(2) = 0.991），在信噪比为3时检测限为0.5 nM。这种新探针相对于传统分子信标对DNA具有更高的选择性。
