使用连接寡核苷酸探针对具有正确插入方向和完整连接的重组DNA克隆进行单步杂交筛选。

L C Au, T Y Lo, H H Wang, K B Choo
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引用次数: 0

摘要

克隆端部为钝端或端部相同的DNA片段,可得到插入序列在两个方向上存在的菌落。阳性克隆的定位和连接序列需要通过酶切分析和/或测序来确定。在这里,我们提出了一种快速的一步筛选方法,不仅具有所需的取向,而且具有完整的连接序列。在该方法中,根据预期的载体插入连接序列合成的16-18 meric寡核苷酸探针作为探针进行集落杂交筛选。使用这种策略,只有符合上述标准的重组克隆才会在杂交中显示出阳性信号。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Single-step hybridization screening for recombinant DNA clones with correct insert orientation and intact junction using a junctional oligonucleotide probe.

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.

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