Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.
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DOI：
10.1021/bm901387t
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发表时间：
2010-04-12
影响因子：
6.2
通讯作者：
Chilkoti, Ashutosh
中科院分区：
文献类型：
--
作者：
McDaniel, Jonathan R.;MacKay, J. Andrew;Quiroz, Felipe Garcia;Chilkoti, Ashutosh
本文报道了一种新的策略，递归定向连接质粒重建（PRe-RDL），快速克隆高重复多肽的任何序列和指定长度在大分子量范围内。在PRe-RDL的单个循环中，亲本质粒的两个半部分（每个半部分包含一个低聚物的拷贝）被连接在一起，从而使低聚物二聚并重新构成一个功能质粒。该过程递归地进行，以组装具有所需重复次数的低聚基因。由于使用了ii型限制性内切酶，PRe-RDL具有几个独特的特征：首先，PRe-RDL是一种无缝克隆方法，在连接连接处没有多余的核苷酸。由于PRe-RDL使用ii型内切酶连接质粒的两个半部分，因此它也解决了RDL的主要限制，即它消除了对可以寡聚的基因序列的任何限制。在PRe-RDL中，只有在成功连接后才能重建功能质粒，这也解决了RDL的另外两个限制：在RDL中观察到的载体自连接的重要背景，以及由于插入物的非生产性循环而导致的连接效率降低。PRe-RDL还可用于组装编码不同序列的基因，以预定顺序编码嵌段共聚物或将前导和后导肽序列附加到寡聚基因上。
This paper reports a new strategy, Recursive Directional Ligation by Plasmid Reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.
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影响因子：
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作者：
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