The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program () for the selection of suitable Body Double enzymes and the design of the appropriate primers.
展开▼
机译：此处描述的过程允许克隆包含限制性内切酶（IIP型）识别位点的PCR片段，该片段用于克隆插入序列。 IIS型核酸内切酶-IIP型酶的双倍体-用于产生相同的突出回文。因此，可以将插入物克隆到载体的IIP型位点，而无需用相同的IIP型酶消化PCR产物。我们通过在PCR引物中掺入IIS类型限制酶的识别位点来实现此目的，该酶在其识别位点之外切割DNA，以使切割位置跨所需的突出端序列。 Body Double消化PCR产物会产生所需的突出端。迄今为止，在克隆反应中使用IIS型限制酶仅用于特殊应用，此处介绍的方法使IIS型酶与IIP酶一样有用，可用于常规克隆。为了帮助找到Body Double酶，我们总结了可能对Body Double克隆有用的IIS型酶，并创建了一个在线程序（），用于选择合适的Body Double酶和设计合适的引物。
展开▼
