High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler
High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler
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DOI：
10.1093/nar/30.9.e39
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发表时间：
2002-05-01
影响因子：
14.9
通讯作者：
Fox, KR
中科院分区：
文献类型：
--
作者：
Darby, RAJ;Sollogoub, M;Fox, KR
我们已经使用含有分子信标的寡核苷酸来确定分子内DNA双链体、三链体和四链体（四链体）的解链谱。在这些研究中使用的合成寡核苷酸含有荧光团（荧光素）和猝灭剂（甲基红）连接到脱氧核糖或dU的5位。在折叠的DNA结构中，荧光团和猝灭剂非常接近，荧光被猝灭。当结构熔化时，荧光团和猝灭剂分离，并且荧光大幅增加。这些实验在Roche LightCycler中进行;这需要少量材料（通常为4 pmol寡核苷酸），并且可以并行进行32个解链图谱。我们已经使用这种技术来比较含有不同的碱基类似物的三链体的稳定性，并确认三链体，而不是双链体，DNA的三链体结合配体的选择性。我们还比较了分子间和分子内四链体的熔化。
We have used oligonucleotides containing molecular beacons to determine melting profiles for intramolecular DNA duplexes, triplexes and quadruplexes (tetraplexes). The synthetic oligonucleotides used in these studies contain a fluorophore (fluorescein) and quencher (methyl red) attached either to deoxyribose or to the 5 position of dU. In the folded DNA structures the fluorophore and quencher are in close proximity and the fluorescence is quenched. When the structures melt, the fluorophore and quencher are separated and there is a large increase in fluorescence. These experiments were performed in a Roche LightCycler; this requires small amounts of material (typically 4 pmol oligonucleotide) and can perform 32 melting profiles in parallel. We have used this technique to compare the stability of triplexes containing different base analogues and to confirm the selectivity of a triplex-binding ligand for triplex, rather than duplex, DNA. We have also compared the melting of inter- and intramolecular quadruplexes.