Hochgeschwender U, Sutcliffe J G, Brennan M B
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8482-6. doi: 10.1073/pnas.86.21.8482.
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.
我们已经建立了一种用于构建特定小鼠染色体文库的方案。通过在特异性阻断仓鼠重复序列后，用放射性标记的小鼠重复序列进行筛选，从仅含有一条小鼠染色体的仓鼠 - 小鼠体细胞杂种的基因组文库中鉴定出含小鼠DNA的克隆；95%的含小鼠DNA的克隆被鉴定出来。我们已将此方案应用于构建小鼠16号染色体文库，该文库由14,200个克隆或两个“染色体当量”组成。每个克隆占据微量滴定板中的一个单独孔，使得整个文库能够被重复且可重复地铺板，并通过杂交进行分析。此外，我们还建立了一种方案，用于制备特异性去除高度重复序列的cDNA探针，以探测基因组克隆文库。通过用来自小鼠肝脏和大脑的cDNA探针筛选16号染色体文库，我们证明了鉴定表达序列并表征其表达模式的可行性。这种染色体特异性文库有助于分离特定的基因座，并为构建整个染色体的综合转录、遗传和物理图谱奠定基础。
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