一种使用混合PCR筛选技术高效延伸水稻BAC-重叠群的方法(英文)  

A High Efficient Approach Used for BAC-contig Extension of Oryza sativa with PCR Screening the BAC Clone Pools

作　　者：胡昊[1] 李滔 穆洁[1] 韩斌[1] 洪国藩[1] 

机构地区：[1]中国科学院上海生命科学研究院国家基因研究中心,上海200233

出　　处：《生物化学与生物物理学报》2002年第3期358-364,共7页

基　　金：水稻基因组测序国际合作项目 (IRGSP );中国科学院资助项目;国家科技部资助项目;上海市科委资助项目~~

摘　　要：使用“克隆连克隆 (clonebyclone)”战略进行水稻基因组测序需要依赖于构建好的基因组物理图。工作着眼于水稻籼稻广陆矮 4号 (OryzasativaindicaGuangLuAi4)第四号染色体长臂上 5 6 .1～ 6 8cM的区域 ,采用PCR方法筛选BAC全库来延伸重叠群 ,构建物理图。通过参照特异遗传探针定位的BAC克隆 (seedBAC)末端序列设计了 14对引物 ,按特定规则分成 3组 ,分别以代表水稻BAC库 (共 2 2 36 8个BAC )的 2 33个BACpool为模板进行PCR反应 ,一共获得了 6 5个阳性BAC克隆 ,通过末端测序、酶切杂交等方法确定了其中 2 9个BAC克隆作为有效延伸的克隆 ,延伸了 8个重叠群。通过酶切杂交、末端测序等方法还获知阳性BAC的延伸方向、延伸长度以及与seedBAC之间的重叠长度。8个重叠群总的延伸长度达到5 10kb。与实验室原用于作物理图的其他方法如指纹图、点杂交等相比 ,该方法有高效率、高灵敏度、专一性好、可重复使用等优点。To extend 8 BAC contigs, which were previously located in the 56.1—68 cM region of the chromosome 4 of the Oryza sativa indica GuangLuAi4, 14 pairs of primers were designed according to the terminal sequences of the existing seed BACs and were deliberately divided into 3 groups. With the 3 groups of primer mixtures, 233 pools of BAC DNA that represent 22 368 BAC clones from O.sativa indica GuangLuAi4 genomic library were screened. 65 positive clones corresponding to the 8 contigs were isolated and 29 clones of them were confirmed to be extended to the seed BACs by end-sequencing and fingerprinting. The protocol greatly enhanced the efficiency of the contig extension and was also superior for its specificity, sensitivity and reusability to the colony in situ hybridization which is a conventional method employed in contig extension and physical map construction.

关 键 词：混合PCR 筛选 高效延伸 水稻 细菌人工染色体 重叠群 

分 类 号：Q78[生物学—分子生物学]