Combing of genomic DNA from droplets containing picograms of material.
Combing of genomic DNA from droplets containing picograms of material.
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DOI：
10.1021/nn5063497
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发表时间：
2015-01-27
期刊：
影响因子：
17.1
通讯作者：
Neely, Robert K.
中科院分区：
文献类型：
--
作者：
Deen, Jochem;Sempels, Wouter;De Dier, Raf;Vermant, Jan;Dedecker, Peter;Hofkens, Johan;Neely, Robert K.
关键词：
线性DNA分子的沉积是许多单分子基因组方法的关键步骤，包括DNA作图、纤维- fish和一些新兴的测序技术。在理想的情况下，为这些实验而沉积的DNA绝对是线性的，并且均匀拉伸，从而能够精确地测量距离。然而，这种情况很少发生，而且，目前在表面上捕获和线性化DNA的方法往往需要复杂的表面制备和大量的起始材料来实现基因组尺度的制图。这使得它们在技术上要求很高，并阻碍了它们在基因组学新兴领域的应用，比如基于单细胞的分析。在这里，我们描述了一种简单而极其有效的方法来沉积和线性化基因组DNA分子。我们使用的液滴只有几十皮克的物质，只需用吸管头把它们拖过涂有聚合物的盖子。在本报告中，我们重点介绍了一种特殊的聚合物，Zeonex，它在捕获DNA方面非常有效。我们描述了在Zeonex表面捕获DNA的方法，并发现使用液滴极大地促进了DNA的有效沉积。这是液滴中循环流动的结果，在表面/溶液的界面处保持了较高的DNA浓度。总的来说，我们的方法为研究基因组结构变异提供了一条可行的途径，这些变异来自不超过少数细胞的样本。
Deposition of linear DNA molecules is a critical step in many single-molecule genomic approaches including DNA mapping, fiber-FISH, and several emerging sequencing technologies. In the ideal situation, the DNA that is deposited for these experiments is absolutely linear and uniformly stretched, thereby enabling accurate distance measurements. However, this is rarely the case, and furthermore, current approaches for the capture and linearization of DNA on a surface tend to require complex surface preparation and large amounts of starting material to achieve genomic-scale mapping. This makes them technically demanding and prevents their application in emerging fields of genomics, such as single-cell based analyses. Here we describe a simple and extremely efficient approach to the deposition and linearization of genomic DNA molecules. We employ droplets containing as little as tens of picograms of material and simply drag them, using a pipet tip, over a polymer-coated coverslip. In this report we highlight one particular polymer, Zeonex, which is remarkably efficient at capturing DNA. We characterize the method of DNA capture on the Zeonex surface and find that the use of droplets greatly facilitates the efficient deposition of DNA. This is the result of a circulating flow in the droplet that maintains a high DNA concentration at the interface of the surface/solution. Overall, our approach provides an accessible route to the study of genomic structural variation from samples containing no more than a handful of cells.
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DOI：
10.1073/pnas.95.14.8046
发表时间：
1998-07-07
影响因子：
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作者：
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通讯作者：
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