DNA cloning without restriction enzyme and ligase.
DNA cloning without restriction enzyme and ligase.
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DOI：
10.2144/99276rr02
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发表时间：
1999-12
期刊：
影响因子：
2.7
通讯作者：
H. Tseng
中科院分区：
文献类型：
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作者：
H. Tseng
使用传统DNA克隆程序的一个常见问题是，对于给定的任务，通常无法获得合适的天然限制性位点。创建新的限制性位点通常是耗时的。在这里，我描述了一个简单的技术，生产“定制的粘性末端”的PCR引物设计和λ核酸外切酶消化的组合。这些互补的粘性末端可以形成杂交体以连接两个序列。由于λ核酸外切酶产生的突出端比互补序列稍长，在杂交体形成后，单链缺口的延伸保留，然后由Klenow（3 '->5'外切-）酶修复。修复过程也稳定了连接。由于不依赖于天然或人工限制性位点，该方法允许将一个DNA片段在几乎任何位置快速和精确地插入另一个DNA片段。它还简化了克隆策略的规划，增加了重组频率，并且适合于自动化。
One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation.