Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level -: art. no. e125
Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level -: art. no. e125
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DOI：
10.1093/nar/gng125
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发表时间：
2003-10-15
影响因子：
14.9
通讯作者：
Escudé, C
中科院分区：
文献类型：
--
作者：
Géron-Landre, B;Roulon, T;Escudé, C
通过将标记的dsDNA片段连接到茎环三聚体形成的寡核苷酸（TFO）上，实现了短序列双链DNA （dsDNA）的荧光标记。TFO通过配体诱导的三螺旋结构缠绕在目标序列上后，其末端相互杂交，留下一个悬垂的单链序列，然后使用T4 DNA连接酶将其连接到荧光dsDNA片段上。在DNA梳理后，用荧光显微镜标记和显示lambda DNA上不重复的15bp序列。该标签位于距离分子一端4.2 +/- 0.5 kb的特定位置，与三螺旋形成目标序列的位置（距离一端4.4 kb）一致。此外，还注意到另一种梳理过程，其中DNA分子从标签上附着到梳理幻灯片上，而不是从其一端附着。本文所描述的方法为检测极短的dsDNA序列提供了一种新的工具，并为单个DNA分子的微操作提供了多种视角。
Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem-loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 +/- 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.