National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.
Plant Methods. 2011 Oct 11;7:33. doi: 10.1186/1746-4811-7-33.
Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors.
The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn.
The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.
大片段插入 BAC 和 BIBAC 文库是真核生物基因组结构和功能基因组学研究的重要工具。为了促进 BAC 和 BIBAC 文库的构建以及完整大片段 BAC 插入物向 BIBAC 载体的转移（这在定位克隆中是期望的），我们开发了一对新的 BAC 和 BIBAC 载体。
新型 BAC 载体 pIndigoBAC536-S 和新型 BIBAC 载体 BIBAC-S 具有以下特点：1）两者均在位置上含有两个反向取向的 18 个碱基对非回文 I-SceI 位点，该位置侧翼有一个包含 lacZ 选择标记和克隆位点的相同 DNA 片段。通过 I-SceI 切割，大 DNA 插入物可以从载体中作为单个片段切除，从而容易对插入物进行大小测定。更重要的是，由于两个载体包含用于转化体选择的不同抗生素抗性基因，并且通过 I-SceI 产生相同的非互补 3'突出的 ATAA 末端，从而抑制自连接和互连接，因此 BAC 和 BIBAC 载体之间完整的大片段基因组 DNA 插入物的交换是直接的；2）两者均被构建为高拷贝复合载体。可分别从高拷贝复合载体 pHZAUBAC1 和 pHZAUBIBAC1 制备可靠的线性化和去磷酸化原始低拷贝 pIndigoBAC536-S 和 BIBAC-S 载体，这些载体可用于文库构建，而无需额外的准备步骤或特殊试剂，从而简化了 BAC 和 BIBAC 文库的构建。使用新型 BIBAC-S 载体构建的 BIBAC 克隆在大肠杆菌和农杆菌中均稳定。可以通过我们的网站 http://GResource.hzau.edu.cn 访问这些载体。
这两个新载体及其各自的高拷贝复合载体可以极大地促进 BAC 和 BIBAC 文库的构建和表征。从一个载体向另一个载体转移完整的大片段基因组 DNA 插入物变得直接。
