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稳态剪切流中的DNA片段化

DNA fragmentation in a steady shear flow.

作者信息

Qiao Yiming, Ma Zixue, Onyango Clive, Cheng Xiang, Dorfman Kevin D

机构信息

Department of Chemical Engineering and Materials Science, University of Minnesota, Twin Cities, 421 Washington Ave SE, Minneapolis, Minnesota 55455, USA.

出版信息

Biomicrofluidics. 2022 Oct 27;16(5):054109. doi: 10.1063/5.0109361. eCollection 2022 Sep.

Abstract

We have determined the susceptibility of T4 DNA (166 kilobase pairs, kbp) to fragmentation under steady shear in a cone-and-plate rheometer. After shearing for at least 30 min at a shear rate of , corresponding to a Reynolds number of and a Weissenberg number of , % of the sample is broken into a polydisperse mixture with a number-averaged molecular weight of  kbp and a polydispersity index of , as measured by pulsed-field gel electrophoresis (with a 95% confidence interval). The molecular weight distributions observed here from a shear flow are similar to those produced by a (dominantly extensional) sink flow of DNA and are qualitatively different than the midpoint scission observed in simple extensional flow. Given the inability of shear flow to produce a sharp coil-stretch transition, the data presented here support a model where polymers can be fragmented in flow without complete extension. These results further indicate that DNA fragmentation by shear is unlikely to be a significant issue in microfluidic devices, and anomalous molecular weight observations in experiments are due to DNA processing prior to observation in the device.

摘要

我们已经测定了T4 DNA(166千碱基对,kbp)在锥板流变仪中稳定剪切作用下的碎片化敏感性。在剪切速率为 下剪切至少30分钟后,对应雷诺数为 、魏森贝格数为 ,通过脉冲场凝胶电泳测量(95%置信区间), %的样品被破碎成多分散混合物,其数均分子量为 kbp,多分散指数为 。此处观察到的剪切流产生的分子量分布与DNA的(主要是拉伸性的)汇流产生的分子量分布相似,并且在性质上与简单拉伸流中观察到的中点断裂不同。鉴于剪切流无法产生尖锐的线圈-拉伸转变,此处呈现的数据支持一种模型,即聚合物在流动中可以碎片化而无需完全伸展。这些结果进一步表明,在微流体装置中,剪切导致的DNA碎片化不太可能是一个重大问题,并且实验中异常的分子量观察结果是由于在装置中观察之前的DNA处理所致。

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