Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay.
Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay.
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DOI：
10.3791/1689
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发表时间：
2010-03-23
期刊：
影响因子：
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通讯作者：
van Oijen, Antoine M
中科院分区：
文献类型：
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作者：
Kulczyk, Arkadiusz W;Tanner, Nathan A;van Oijen, Antoine M
我们描述了一种观察由噬菌体复制系统的蛋白质介导的单个DNA分子实时复制的方法。线性化的DNA被修饰成在一条链的末端有一个生物素，在同一条链的另一端有一个地高辛苷片段。生物素化的端连接到功能化的玻璃盖盖上，二氧原化的端连接到小珠上。这些dna珠系在流动池表面的组装允许层流施加在珠上的阻力。结果，DNA被拉伸到靠近并平行于盖盖表面，其力由流速决定（图1）。DNA的长度是通过监测珠子的位置来测量的。利用单链和双链DNA之间的长度差异来获得叉上复制蛋白活性的实时信息。测量头的位置可以精确地确定DNA解绕和聚合的速率和过程（图2）。
We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized lambda DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).