Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point.
Single-molecule sequence detection via microfluidic planar extensional flow at a stagnation point.
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DOI：
10.1039/b926847b
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发表时间：
2010-06-21
期刊：
影响因子：
6.1
通讯作者：
Muller SJ
中科院分区：
文献类型：
--
作者：
Dylla-Spears R;Townsend JE;Jen-Jacobson L;Sohn LL;Muller SJ
我们展示了使用微流体停滞点流来捕获和延伸双链（ds）基因组DNA的单分子，用于检测沿着DNA骨架的靶序列。基于EcoRI的突变荧光标记物与荧光标记的ds λ-DNA序列特异性结合。将标记物-DNA复合物引入由以90度相交的流动通道组成的微流体交叉槽中。含有标记物-DNA复合物的缓冲溶液在交叉槽的一个通道中流动，纯缓冲液以相同的流速在相对的通道中流动，并且流体从入口通道以九十度离开两个通道。这在平面延伸流的中心处产生停滞点，其中标记物-DNA复合物可被捕获并沿流出轴沿着伸长。可以使用流动强度（即，无量纲流速）。使用荧光显微镜直接观察DNA骨架和沿着拉伸DNA结合的标记物，并测量标记物沿着DNA骨架的位置。我们发现，我们的方法允许检测的五个预期的靶位点位置的每一个在1.5 kb的标准偏差<1.5 kb。我们将该方法在轮廓长度68%和88%的分子延伸处的精确度和准确度与通过分子梳理获得的类似数据的结合分布进行了比较。我们还提供了证据表明，在标记物与DNA结合期间增加样品的混合可以改善与dsDNA的内部靶序列的结合，这可能是通过延伸DNA并使内部结合位点更容易接近。
We demonstrate the use of a microfluidic stagnation point flow to trap and extend single molecules of double-stranded (ds) genomic DNA for detection of target sequences along the DNA backbone. Mutant EcoRI-based fluorescent markers are bound sequence-specifically to fluorescently labeled ds λ-DNA. The marker-DNA complexes are introduced into a microfluidic cross slot consisting of flow channels that intersect at ninety degrees. Buffered solution containing the marker-DNA complexes flows in one channel of the cross slot, pure buffer flows in the opposing channel at the same flow rate, and fluid exits the two channels at ninety degrees from the inlet channels. This creates a stagnation point at the center of a planar extensional flow, where marker-DNA complexes may be trapped and elongated along the outflow axis. The degree of elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device. Both the DNA backbone and the markers bound along the stretched DNA are observed directly using fluorescence microscopy and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb. We compare the method’s precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via molecular combing. We also provide evidence that increased mixing of the sample during binding of the marker to the DNA improves binding to internal target sequences of dsDNA, presumably by extending the DNA and making the internal binding sites more accessible.
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影响因子：
7.4
作者：
Dylla-Spears, Rebecca;Townsend, Jacqueline E.;Sohn, Lydia L.;Jen-Jacobson, Linda;Muller, Susan J.
通讯作者：
Muller, Susan J.
影响因子：
8.6
作者：
BENSIMON, D;SIMON, AJ;BENSIMON, A
通讯作者：
BENSIMON, A
DOI：
10.1073/pnas.0401638101
发表时间：
2004-04-27
影响因子：
11.1
作者：
Gordon, MP;Ha, T;Selvin, PR
通讯作者：
Selvin, PR
影响因子：
5.5
作者：
LARSON, RG;MAGDA, JJ
通讯作者：
MAGDA, JJ
影响因子：
14.9
作者：
Géron-Landre, B;Roulon, T;Escudé, C
通讯作者：
Escudé, C