We present a simple and novel assay--employing a universal molecular beacon (MB) in the presence of Hgpo--for the detection of single nucleotide polymorphisms (SNPs) based on Hgpo-DNA complexes inducing a conformational change in the MB. The MB (T-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 Hgpo-T-MB complexes through T-Hgpo-T bonding, the conformation of T-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 Hgpo-T-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-MB, 20 mM NaCl, 1.0 oM Hgpo, 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 (Rpo = 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.
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机译：我们提出了一种简单而新颖的检测方法-在Hgpo存在下使用通用分子信标（MB）-用于检测基于MB诱导MB构象变化的Hgpo-DNA复合物的单核苷酸多态性（SNP）。 MB（T-MB）包含一个19-mer环和一对七个胸苷（T）碱基的茎，一个位于5'端的羧基荧光素（FAM）单元和一个4-（[4-（二甲基氨基）苯基]偶氮）苯甲酸（DABCYL）单元位于3'端。通过T-Hgpo-T键形成Hgpo-T-MB络合物后，T-MB的构象从无规卷曲变为折叠结构，导致FAM和DABCYL单元之间的距离缩短，从而提高了效率FAM和DABCYL单元之间的荧光共振能量转移（FRET）的变化，导致MB的荧光强度降低。在存在互补DNA的情况下，形成了双链DNA复合物（而不是Hgpo-T-MB复合物），与折叠结构相比，FAM和DABCYL单元之间的FRET发生的程度较小。在最佳条件下（20 nM T-MB，20 mM NaCl，1.0 oM Hgpo，5.0 mM磷酸盐缓冲溶液，pH 7.4），荧光强度对完全匹配的DNA浓度的线性图在2- 30 nM（Rpo = 0.991），信噪比为3时检测限为0.5 nM。与常规MB相比，这种新探针对DNA的选择性更高。
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