loxP-directed cloning: Use Cre recombinase as a universal restriction enzyme
loxP-directed cloning: Use Cre recombinase as a universal restriction enzyme
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DOI：
10.2144/01314rr02
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发表时间：
2001-10-01
期刊：
影响因子：
2.7
通讯作者：
Bishop, JM
中科院分区：
文献类型：
--
作者：
Buchholz, F;Bishop, JM
我们开发了一种使用Cre/loxP系统进行体外DNA操作的新方法，以及一种将DNA克隆成环状片段的技术。该方法快速、可靠，并且允许将DNA片段灵活地克隆到含有loxP位点的片段中。我们发现，loxP位点可以作为克隆任何限制性内切酶消化的DNA片段的通用靶位点。该技术通过在载体的所需方向和读框中生成定制设计的5‘,3’或钝端，从而消除了克隆载体和靶标时对兼容限制性位点的需要。因此，该方法消除了将DNA片段克隆到具有有限克隆位点的载体时所遇到的限制。34bp的loxP序列确保了独特性，即使在大片段被操纵时也是如此。我们提出三个例子，包括细菌人工染色体的操作。由于DNA操作发生在loxP位点，因此我们将这种技术称为loxP定向克隆。
We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allows flexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom designed 5', 3', or blunt ends in the desired orientation and reading frame in the vector. Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.