Liu Chi-Wei, Lin Yang-Wei, Huang Chih-Ching, Chang Huan-Tsung
Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
Biosens Bioelectron. 2009 Apr 15;24(8):2541-6. doi: 10.1016/j.bios.2009.01.003. Epub 2009 Jan 14.
We have developed a universal molecular beacon (T(7)-MB-T(7)) for the detection of single-nucleotide polymorphisms (SNPs). The beacon, which contains a 19-mer loop and a stem comprising a pair of seven thymidine (T) bases, forms double-stranded structures with target DNA molecules, leading to increases in the fluorescence of ethidium bromide (EthBr) as a result of intercalation. The interactions of the beacon with perfectly matched (DNA(pm)) and single-base mismatched (DNA(mm)) DNA strands are stronger and weaker, respectively, than those with Hg(2+) ions. As a result, the fluorescence of a solution containing T(7)-MB-T(7), DNA(pm), EthBr, and Hg(2+) is higher than that of a corresponding solution containing T(7)-MB-T(7), DNA(mm), EthBr, and Hg(2+), because the former has a greater number of intercalation sites for EthBr. Under the optimal conditions (100 nM T(7)-MB-T(7), 20 mM NaCl, 5.0 microM Hg(2+), and 300 nM EthBr in 5.0 mM Tris-HCl solution, pH 7.4), the plot of the fluorescence intensity against the concentration of DNA(pm) was linear over the range 5.0-100 nM (R(2)=0.98). A similar probe, T(7)-MB(t)-T(7), is sensitive and selective for the detection of a gene associated with hereditary tyrosinemia type I. Relative to conventional MBs, our new probe offers the advantages of higher selectivity toward DNA, less nonspecific binding toward single-stranded-DNA-binding protein, greater resistance to nuclease digestion, and low cost; therefore, we suspect that this system holds great potential for practical studies of SNPs.
我们开发了一种用于检测单核苷酸多态性（SNP）的通用分子信标（T(7)-MB-T(7)）。该信标包含一个19个核苷酸的环和一个由一对七个胸腺嘧啶（T）碱基组成的茎，与目标DNA分子形成双链结构，由于嵌入作用导致溴化乙锭（EthBr）荧光增强。与汞离子（Hg(2+)）相比，该信标与完全匹配的DNA（DNA(pm)）和单碱基错配的DNA（DNA(mm)）链的相互作用分别更强和更弱。因此，含有T(7)-MB-T(7)、DNA(pm)、EthBr和Hg(2+)的溶液的荧光高于含有T(7)-MB-T(7)、DNA(mm)、EthBr和Hg(2+)的相应溶液，因为前者具有更多的EthBr嵌入位点。在最佳条件下（5.0 mM Tris-HCl溶液，pH 7.4中100 nM T(7)-MB-T(7)、20 mM NaCl、5.0 μM Hg(2+)和300 nM EthBr），荧光强度相对于DNA(pm)浓度的曲线在5.0 - 100 nM范围内呈线性（R(2)=0.98）。一种类似的探针T(7)-MB(t)-T(7)对检测与I型遗传性酪氨酸血症相关的基因敏感且具有选择性。相对于传统分子信标，我们的新探针具有对DNA选择性更高、对单链DNA结合蛋白非特异性结合更少、对核酸酶消化抵抗力更强以及成本低等优点；因此，我们怀疑该系统在SNP的实际研究中具有巨大潜力。
