Stable Positional cloning of long continuous DNA in the Bacillus subtilis genome vector
Stable Positional cloning of long continuous DNA in the Bacillus subtilis genome vector
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DOI：
10.1093/jb/mvg168
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发表时间：
2003-10-01
影响因子：
2.7
通讯作者：
Tsuge, K
中科院分区：
文献类型：
--
作者：
Itaya, M;Fujita, K;Tsuge, K
首次证明了在枯草芽孢杆菌基因组载体中直接克隆长的连续基因组片段。在克隆之前，必须将两个小的DNA片段安装在载体中。通过同源重组克隆这两个片段之间的DNA。使用蓝细菌集胞藻PCC 6803的3，573-kb基因组估计克隆效率。使用芽孢杆菌基因组载体的内部选择系统或使用蓝藻基因组中的抗生素抗性标记来选择澄清剂。通过一个单一的程序克隆指定的基因组片段大至77 kb。克隆效率受供体DNA的分子量和待克隆DNA的大小影响。该方法适用于大尺寸DNA的直接靶向克隆。
Direct cloning of a long continuous genome segment in a Bacillus subtilis genome vector was demonstrated for the first time. Two small DNA fragments had to be installed in the vector prior to cloning. The DNA between these two fragments was cloned via homologous recombination. The efficiency of cloning was estimated using the 3,573-kb genome of a cyanobacterium, Synechocystis sp. PCC 6803. Recombinants were selected using the internal selection system of the Bacillus genome vector or with the antibiotic resistance marker in the cyanobacterial genome. Designated genomic segments as large as 77-kb were cloned by means of a single procedure. Cloning efficiency is affected by the molecular weight of the donor DNA and the size of the DNA to be cloned. The method is suitable for direct target cloning of large-sized DNA.