Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules
Automated high resolution optical mapping using arrayed, fluid-fixed DNA molecules
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DOI：
10.1073/pnas.95.14.8046
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发表时间：
1998-07-07
影响因子：
11.1
通讯作者：
Schwartz, DC
中科院分区：
文献类型：
--
作者：
Jing, JP;Reed, J;Schwartz, DC
新的映射方法构建有序的限制性图谱从荧光显微镜图像的个人，核酸内切酶消化的DNA分子。在光学映射中，分子被拉长并固定在衍生的玻璃表面上，在酶消化后保持生物化学可及性和片段顺序。相对荧光强度和表观长度的测量确定的限制性片段的大小，使有序的地图建设，而无需电泳分析，光学映射系统在这里报道的是基于我们的物理特性的影响，使用微小的，蒸发液滴内开发的流体流动，延长和固定DNA分子到衍生化的表面。这种蒸发驱动的分子固定产生了限制性内切核酸酶，特别是DNA聚合酶I可接近的良好伸长的分子。然后，我们开发了机器人装置，以平行消化和分析的良好定义的阵列中的网格DNA斑点。为了有效地利用这种效应进行高通量基因组作图，我们开发了：（i）机器视觉和自动图像采集技术，用于处理网格化样品中固定的消化分子，以及（ii）贝叶斯推理方法，用于分析机器视觉数据，从单个DNA分子的图像自动产生高分辨率限制性图谱，这项工作的总的意义是一个集成的系统的发展，映射小插入克隆允许生化数据从工程合奏的个别分子自动积累和分析地图建设。这些方法对于使用统计学上有意义的群体大小的个体分子的各种生化分析是足够普遍的。
New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis, The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I, We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high resolution restriction maps from images of individual DNA molecules, The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.