Au L C, Lo T Y, Wang H H, Choo K B
Department of Medical Research, National Yang-Ming University Taipei, Taiwan, Republic of China.
Proc Natl Sci Counc Repub China B. 1996 Apr;20(2):27-30.
Cloning of DNA fragments with blunt ends or identical protruding ends results in colonies with the insertion sequence existing in two possible orientations. The orientation and the junctional sequence of the positive clones need to be established by means of restriction analysis and/or sequencing. Here, we proposed a rapid one-step method for the screening of clones not only with the desired orientation, but also with an intact junctional sequence. In this method, a 16-18 meric oligonucleotide probe synthesized according to the expected vector-insert junctional sequence is used as a probe in colony hybridization screening. Using this strategy, only recombinant clones which fulfill the above criteria will show positive signals in hybridization.
克隆具有平端或相同突出端的DNA片段会导致菌落中插入序列以两种可能的方向存在。阳性克隆的方向和连接序列需要通过限制性分析和/或测序来确定。在此，我们提出了一种快速的一步法，用于筛选不仅具有所需方向，而且具有完整连接序列的克隆。在该方法中，根据预期的载体-插入物连接序列合成的16 - 18聚体寡核苷酸探针用作菌落杂交筛选中的探针。使用该策略，只有符合上述标准的重组克隆在杂交中才会显示阳性信号。
