An efficient cloning of DNA fragments by a method based on uracil-DNA glycosylase and endonuclease IV
An efficient cloning of DNA fragments by a method based on uracil-DNA glycosylase and endonuclease IV
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基于尿嘧啶-DNA 糖基化酶和核酸内切酶 IV 的方法有效克隆 DNA 片段。
DOI：
10.1016/j.jbbm.2007.07.006
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发表时间：
2008-04-24
影响因子：
--
通讯作者：
Liu, Jianhua
中科院分区：
文献类型：
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作者：
Hou, Jingli;Liu, Xipeng;Liu, Jianhua
本文介绍了一种不依赖于连接反应而克隆随机DNA片段的新方法。该方法涉及在PCR扩增DNA上产生长的突出末端。用于扩增载体DNA的两种寡核苷酸在从5'端起的第10个位置处携带一个尿嘧啶残基，这使得通过尿嘧啶-DNA糖基化酶（UDG）和核酸内切酶IV（Endo IV）产生线性化载体的3'突出端成为可能。设计了76组退火的寡核苷酸，其在3 '端突出10个核苷酸，与线性化载体3'端的核苷酸互补。将线性化的载体和退火的寡核苷酸混合在一起以直接转化大肠杆菌而不进行连接反应。在平板上生长的克隆数已被证明达到1 × 10（5）转化体/μ g，96.1%的转化体携带克隆片段。从转化结果可以看出，本方法产生3'端突出末端的效率较高，且本方法有利于重组子的高效、简便地生产。(c)2007 Elsevier B. V.保留所有权利。
We introduced a novel method to clone random DNA fragments independent of ligation reaction. The method involves the generation of long protruding ends on PCR amplification DNA. Both oligonucleotides used for the amplification of the vector DNA carried one uracil residue at the tenth position from the 5' end and this made the creation of the 3' protruding ends of linearized vector possible by uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). 76 groups of annealed oligonucleotides that had ten-nucleotides protruding at 3'-end, which were complementary to those at 3'-end of the linearized vector, were designed. The linearized vector and the annealed oligonucleotide were mixed together to transform E.coli directly without ligation reaction. The number of the clone that grew on the plates had been demonstrated to reach 1 x 10(5) transformants/mu g and 96.1% of transformants harbored the cloned fragments. From the results of transformation, we can confirm that the efficiency of the creation of 3' protruding ends in our method is high and our cloning method is benefit to produce recombinants easily and efficiently. (c) 2007 Elsevier B.V. All rights reserved.