Efficient random subcloning of DNA sheared in a recirculating point-sink flow system
Efficient random subcloning of DNA sheared in a recirculating point-sink flow system
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DOI：
10.1093/nar/24.20.3879
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发表时间：
1996-10-15
影响因子：
14.9
通讯作者：
Davis, RW
中科院分区：
文献类型：
--
作者：
Oefner, PJ;HunickeSmith, SP;Davis, RW
基于高效液相色谱泵，我们已经建立了一种装置，该装置允许DNA通过63 μ m的孔再循环，随后分级分离至~ 300个碱基对的最小片段大小。发现DNA片段在由突然收缩产生的会聚流中的停留时间足够长，以允许DNA分子延伸成高度延伸的构象，因此，在大多数情况下，30次传代足以获得窄的尺寸分布，>90%的片段位于2倍尺寸分布内。发现实现断裂所需的剪切速率与分子量的1.0次方成反比。与限制性消化相比，高达40%的所有片段可以直接克隆，在用Klenow、T4聚合酶或T4多核苷酸激酶进行在先末端修复时，仅观察到克隆效率的边际改进。测序显示片段的分布相当随机。
Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-mu m orifice with ensuing fractionation to a minimum fragment size of -300 base pairs, Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint, In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution, The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight, Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.