Department of Chemical Engineering, University of California, Berkeley, Berkeley, California, USA.
Anal Chem. 2009 Dec 15;81(24):10049-54. doi: 10.1021/ac9019895.
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.
我们利用突变的 EcoRI 限制内切酶（K249C）创建了一种荧光标记物，该标记物能够长时间直接可视化双链 (ds) DNA 上特定序列的基因组长度。该标记物由一个生物素化酶组成，通过生物素-亲和素相互作用连接到荧光纳米球上。通过在突变位点处生物素化突变型 EcoRI，实现了对酶结合口袋中生物素位置的控制。将生物素化酶与 dsDNA 和中性亲和素包被的荧光纳米球一起孵育，在允许酶结合但防止切割的条件下进行孵育。然后用荧光染料对标记有 DNA 的 DNA 进行染色，并在聚赖氨酸包被的载玻片上拉伸，以便可以测量单个 DNA 分子上结合标记物的位置。我们使用批量凝胶迁移分析和单分子方法证明了标记物特异性结合其靶序列的能力。
