Oefner P J, Hunicke-Smith S P, Chiang L, Dietrich F, Mulligan J, Davis R W
Department of Biochemistry, Stanford University, CA, USA.
Nucleic Acids Res. 1996 Oct 15;24(20):3879-86. doi: 10.1093/nar/24.20.3879.
Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 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.
基于一台高效液相色谱泵，我们构建了一种装置，该装置可使DNA通过一个63微米的小孔再循环，随后进行分级分离，得到最小片段大小约为300个碱基对的片段。发现DNA片段在由突然收缩产生的汇聚流中的停留时间足够长，足以使DNA分子延伸成高度伸展的构象，从而在中点处发生断裂。在大多数情况下，30次通过就足以获得窄的大小分布，超过90%的片段位于2倍大小分布范围内。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。与限制性酶切消化相比，所有片段中高达40%可以直接克隆，在用Klenow酶、T4聚合酶或T4多核苷酸激酶进行末端修复后，克隆效率仅有微小提高。测序显示片段分布相当随机。
