Single DNA molecule stretching in sudden mixed shear and elongational microflows
Single DNA molecule stretching in sudden mixed shear and elongational microflows
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DOI：
10.1039/b602845d
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发表时间：
2006-01-01
期刊：
影响因子：
6.1
通讯作者：
Gilmanshin, Rudolf
中科院分区：
文献类型：
--
作者：
Larson, Jonathan W.;Yantz, Gregory R.;Gilmanshin, Rudolf
在连续延伸流中对单个DNA分子的高通量拉伸和监测为生物技术应用如DNA作图提供了令人信服的优势。然而，在普通微流体实施方式中的聚合物动力学通常由于剪切相互作用而复杂化。通过观察荧光标记的185 kb细菌人工染色体在漏斗状微流体通道中产生的突然混合剪切和伸长微流中，研究了这些效应。单个游离DNA分子的延伸作为累积流体应变和应变速率的函数进行了研究。在恒定或逐渐变化的应变速率条件下，通过突然拉伸分量的拉伸如先前对于理想拉伸流动（T. T. Perkins，D. E. Smith和S. Chu，Science，1997，276，2016）：首先，增加的累积流体应变和增加的应变速率产生更高的拉伸效率，尽管剪切相互作用的复杂性;其次，结果与主要处于发夹构象的未拉伸分子一致。更突然的应变速率曲线并没有提供一个均匀的人口高度延伸的分子，突出了剪切和拉伸组件之间的平衡在微流体环境中的DNA拉伸应用的重要性。DNA大小与高达10%的分辨率被证明。总的来说，该装置以与衍射限制的光学序列基序映射兼容的方法每分钟递送1000个拉伸的DNA分子，并且不需要对DNA或芯片表面进行费力的化学修饰。因此，该方法特别适用于DNA混合物的遗传表征，例如在高水平背景DNA中的病原体指纹图谱。
High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.