Ito T, Ohgusu H, Oishi N, Sakaki Y
Department of Preventive Medicine, Nagasaki University, Japan.
Genomics. 1991 Apr;9(4):707-12. doi: 10.1016/0888-7543(91)90364-k.
We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.
我们之前开发了一种高效的程序，用于选择性克隆稀有切割连接片段，该程序基于通过脉冲场聚丙烯酰胺凝胶电泳（PF-PAGE）对连接克隆DNA进行物理分离。与传统的基于克隆的方法相比，物理选择程序的一个优点是，传统方法利用将选择标记或载体序列插入稀有切割位点，而物理选择程序可以很容易地应用于选择产生模糊粘性末端序列（如SfiI（GGCCNNNN/NGGCC））的稀有切割连接片段。在本研究中，通过在PF-PAGE中引入不连续缓冲系统改进了物理分离程序，并通过从人21号染色体特异性文库中选择性分离SfiI连接克隆证明了其可行性。这种简单有效的程序将为基因组分析提供一个有用的工具。
