Fluorescent marker for direct detection of specific dsDNA sequences.
Fluorescent marker for direct detection of specific dsDNA sequences.
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DOI：
10.1021/ac9019895
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发表时间：
2009-12-15
影响因子：
7.4
通讯作者：
Muller, Susan J.
中科院分区：
文献类型：
--
作者：
Dylla-Spears, Rebecca;Townsend, Jacqueline E.;Sohn, Lydia L.;Jen-Jacobson, Linda;Muller, Susan J.
我们使用突变EcoRI限制性内切酶（K249C）创建了一种荧光标记，可以长时间、直接地显示双链DNA基因组长度上的特定序列。该标记由生物素化酶组成，通过生物素-亲和素相互作用附着在荧光纳米球上。通过在突变位点对突变体EcoRI进行生物素化，可以控制该酶结合袋的生物素位置。生物素化酶与dsDNA和neutravidin包被的荧光纳米球在允许酶结合但防止裂解的条件下孵育。然后在聚赖氨酸涂层的玻片上荧光染色和拉伸带有标记的DNA，这样就可以测量单个DNA分子上结合的标记的位置。我们证明了标记物的能力，具体结合到其目标序列使用散装凝胶移位测定和单分子方法。
We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme’s binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker’s ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.
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