Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system
Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system
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DOI：
10.1016/j.jbiotec.2003.10.001
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发表时间：
2004-02-05
影响因子：
4.1
通讯作者：
Imamoto, F
中科院分区：
文献类型：
--
作者：
Sasaki, Y;Sone, T;Imamoto, F
研究了Multisite Gateway克隆系统的六种类型的重组信号DNA序列在LR和BP重组反应中的特异性和效率。在通过分别包含在Entry克隆和Destination载体中的attL和attR信号之间的重组产生表达克隆的LR反应中，检查了各种attL和attR对对六种类型的相应信号序列的交叉反应性。在BP反应中，通过将表达克隆或attB侧翼PCR产物中的靶DNA片段转移到供体载体中来创建Entry克隆。测试attB和attP对的各种组合在重组中的反应性。所得结果表明，仅使用匹配的att信号对（例如attL 3-attR 3），交叉反应的特异性和效率显着更高。attB 5-attP 5等，从而验证了在Gateway系统中高通量产生阳性克隆，其中多个重组信号一起存在于一个反应系统中。显示了在质粒中快速构建三或四个DNA融合结构的实例。(C)2003 Elsevier B. V.保留所有权利。
Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector. various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3. attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown. (C) 2003 Elsevier B.V. All rights reserved.