Restriction digestion monitors facilitate plasmid construction and PCR cloning
Restriction digestion monitors facilitate plasmid construction and PCR cloning
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DOI：
10.2144/04366st03
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发表时间：
2004-06-01
期刊：
影响因子：
2.7
通讯作者：
Lowry, C
中科院分区：
文献类型：
--
作者：
Anand, RD;Sertil, O;Lowry, C
通过用两种不同的限制性内切酶消化的片段的“强制”或“定向”连接来构建质粒是高效的，除了当一个位点的抑制消化有利于载体再环化时。这样的失败通常是因为在载体多聚接头或PCR片段的末端克隆位点未检测到不完全的双重消化。为了间接测试切割效率，将“监测”质粒添加到消化物中。在合适的监测器中，两个检测位点被足够的DNA（约全长的20%）分开，以区分双重消化物和封闭的单一消化物。为了使其适用于32种常用克隆酶的组合，我们构建了一组4个监测器（pDM1、pDM2、pDM3和pDM4）。每个含有三个多聚接头，由约1 kb的填充片段隔开。32个位点分布在多聚接头中，使得组中至少一个质粒对于每个酶对是诊断性的。该套件的设计可扩展至多达81个站点。监测器的线性化版本允许确定两种酶中的哪一种在不完全双重消化中失败，并且当靶DNA接近pDM主链的大小时也是有用的。这些质粒还可作为任何位点组合的通用自我监测克隆载体。
Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCRfragnient. To test cleavage efficiency indirectly, a "monitor" plasmid is added to the digest. In a suitable monitor the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the Jailed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows,for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.