Construction and screening of a genomic library specific for mouse chromosome 16.
Construction and screening of a genomic library specific for mouse chromosome 16.
复制标题
小鼠16号染色体特异性基因组文库的构建和筛选。
DOI：
10.1073/pnas.86.21.8482
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发表时间：
1989
影响因子：
11.1
通讯作者：
Brennan,MB
中科院分区：
文献类型：
--
作者：
Hochgeschwender,U;Sutcliffe,JG;Brennan,MB
我们已经建立了一个制作特定小鼠染色体文库的协议。从只含一条小鼠染色体的仓鼠-小鼠体细胞杂交基因组文库中，通过特异性阻断仓鼠重复序列，用放射性标记的小鼠重复序列进行筛选，鉴定出含小鼠dna的克隆；95%的含dna的小鼠克隆被鉴定出来。我们已经应用这个方法建立了一个小鼠16号染色体文库，包含14200个克隆或两个“染色体等同”。每个克隆在微滴盘中占据一个单独的孔，允许整个文库重复和可重复地镀和杂交分析。此外，我们已经建立了一种方案，使cDNA探针特异性地去除高重复序列，用于探测基因组克隆文库。利用小鼠肝脏和大脑的cDNA探针筛选16号染色体文库，证明了鉴定表达序列和表征其表达模式的可行性。这样的染色体特异性文库可以促进已定义的遗传位点的分离，并为整个染色体的综合转录、遗传和物理图谱的生成奠定基础。
We have established a protocol for producing libraries of specific mouse chromosomes. The mouse DNA-containing clones from a genomic library of a hamster-mouse somatic cell hybrid containing only one mouse chromosome are identified by screening with radiolabeled mouse repetitive sequences after specifically blocking hamster repetitive sequences; 95% of the mouse DNA-containing clones are identified. We have applied this protocol in producing a library of mouse chromosome 16, consisting of 14,200 clones or two "chromosome equivalents." Each clone occupies an individual well in a microtiter tray, allowing the entire library to be repeatedly and reproducibly plated and analyzed by hybridization. Further, we have established a protocol for making cDNA probes specifically depleted of highly repetitive sequences for probing libraries of genomic clones. By screening the chromosome 16 library with cDNA probes from mouse liver and brain, we demonstrate the feasibility of identifying expressed sequences and characterizing their patterns of expression. Such chromosome-specific libraries can facilitate the isolation of defined genetic loci as well as form the basis for the production of integrated transcriptional, genetic, and physical maps of entire chromosomes.