Sasaki Yukari, Sone Takefumi, Yoshida Shouhei, Yahata Kazuhide, Hotta Junko, Chesnut Jonathan D, Honda Takeshi, Imamoto Fumio
Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita, Osaka 565-0871, Japan.
J Biotechnol. 2004 Feb 5;107(3):233-43. doi: 10.1016/j.jbiotec.2003.10.001.
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.
对多位点Gateway克隆系统的六种重组信号DNA序列在LR和BP重组反应中的特异性和效率进行了研究。在通过分别包含在入门克隆和目的载体中的attL和attR信号之间的重组来生成表达克隆的LR反应中，检测了六种各自信号序列上各种attL和attR对的交叉反应性。在通过将表达克隆中的目标DNA片段或attB侧翼的PCR产物转移到供体载体中来创建入门克隆的BP反应中，测试了attB和attP对的各种组合在重组中的反应性。获得的结果表明，与不匹配的信号对（如attL3-attR5、attB5-attP3等）相比，只有匹配的att信号对（如attL3-attR3、attB5-attP5等）具有明显更高的交叉反应特异性和效率，从而验证了在一个反应系统中存在多个重组信号的Gateway系统中阳性克隆的高通量生产。展示了在质粒中快速构建三或四个DNA融合结构的实例。
