Grass carp （Ctenopharyngodon idellus） genomic fosmid library cotaining 129 014 clones was constructed and characterized from one diploid grass carp. The average insert size of the fosmid library was determined to be...Grass carp （Ctenopharyngodon idellus） genomic fosmid library cotaining 129 014 clones was constructed and characterized from one diploid grass carp. The average insert size of the fosmid library was determined to be 35 kb by pulsed field gel electrophoresis, which is 4.1-fold coverage of the grass carp genome. To demonstrate the probability of picking the functional genes from the library, eleven functional genes were screened by three-dimensional PCR technique. The number of positive clones of these genes was from 1 to 6. So, this library may screen any useful genes from grass carp. This grass carp genome fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the grass carp genome.展开更多
As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome （BAC） by BAC strategy. The current physical contigs are expec...As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome （BAC） by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.展开更多
利用双重杂交筛选法从已经构建的 A 型新生隐球菌 DNA 克隆库(pCN)中筛选特异性探针。依据克隆库载体 pUC18的核苷酸序列设计并合成一对引物,PCR 扩增克隆库的插入片段,制备杂交膜。同位素标记26种鉴别菌及正常人的总 DNA 与克隆库的 P...利用双重杂交筛选法从已经构建的 A 型新生隐球菌 DNA 克隆库(pCN)中筛选特异性探针。依据克隆库载体 pUC18的核苷酸序列设计并合成一对引物,PCR 扩增克隆库的插入片段,制备杂交膜。同位素标记26种鉴别菌及正常人的总 DNA 与克隆库的 PCR 产物杂交,获得28个候选克隆。同位素标记候选克隆的插入片段(以低熔点胶回收)与上述27种鉴别 DNA 杂交,获得特异性探针。结果:从已经构建的 pCN 中筛选出了3个特异性探针:pCNⅡA6、pCNⅡB5、pCNⅢG1,其中 pCNⅡA6只与A型新生隐球菌的 DNA 杂交,而不与其它鉴别 DNA 杂交,为 A 型特异性克隆;pCNⅡB5只与新生隐球菌的 DNA 杂交,为种特异性克隆;pCNⅢG1只与 A、D 型新生隐球菌的 DNA 杂交,为新生变种特异性克隆。应用上述特异性探针可以对隐球菌感染进行早期快速诊断及区分新生变种和格特变种。展开更多
基金supported by the National Natural Science Foundation of China (30371 098)
文摘Grass carp （Ctenopharyngodon idellus） genomic fosmid library cotaining 129 014 clones was constructed and characterized from one diploid grass carp. The average insert size of the fosmid library was determined to be 35 kb by pulsed field gel electrophoresis, which is 4.1-fold coverage of the grass carp genome. To demonstrate the probability of picking the functional genes from the library, eleven functional genes were screened by three-dimensional PCR technique. The number of positive clones of these genes was from 1 to 6. So, this library may screen any useful genes from grass carp. This grass carp genome fosmid library will be integrated in the presently ongoing efforts to determine the sequence of the grass carp genome.
基金This work was supported by grants from the National Academy of Agricultural Science(Code #200901FHT020508369)the BioGreen21 Program(Code #20050301034438 and Code #20070301034037),Rural Development Administration, Republic of Korea
文摘As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome （BAC） by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.
文摘利用双重杂交筛选法从已经构建的 A 型新生隐球菌 DNA 克隆库(pCN)中筛选特异性探针。依据克隆库载体 pUC18的核苷酸序列设计并合成一对引物,PCR 扩增克隆库的插入片段,制备杂交膜。同位素标记26种鉴别菌及正常人的总 DNA 与克隆库的 PCR 产物杂交,获得28个候选克隆。同位素标记候选克隆的插入片段(以低熔点胶回收)与上述27种鉴别 DNA 杂交,获得特异性探针。结果:从已经构建的 pCN 中筛选出了3个特异性探针:pCNⅡA6、pCNⅡB5、pCNⅢG1,其中 pCNⅡA6只与A型新生隐球菌的 DNA 杂交,而不与其它鉴别 DNA 杂交,为 A 型特异性克隆;pCNⅡB5只与新生隐球菌的 DNA 杂交,为种特异性克隆;pCNⅢG1只与 A、D 型新生隐球菌的 DNA 杂交,为新生变种特异性克隆。应用上述特异性探针可以对隐球菌感染进行早期快速诊断及区分新生变种和格特变种。
