Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.
Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.
复制标题
10 倍基因组等效大鼠 P1 衍生人工染色体文库的构建和表征。
DOI：
10.1006/geno.1998.5319
复制
发表时间：
1998
期刊：
影响因子：
4.4
通讯作者：
deJong,PJ
中科院分区：
文献类型：
--
作者：
Woon,PY;Osoegawa,K;Kaisaki,PJ;Zhao,B;Catanese,JJ;Gauguier,D;Cox,R;Levy,ER;Lathrop,GM;Monaco,AP;deJong,PJ
大鼠 PAC 文库是在载体 pPAC4 中用从雌性棕色挪威大鼠分离的基因组 DNA 构建的。该文库由排列在 614 384 孔微量滴定板中的 215,409 个克隆组成。根据 217 个随机分离的克隆估计平均插入大小为 143 kb，因此代表大约 10 倍的基因组覆盖度。这种覆盖度提供了文库在基因组筛选中包含独特序列的可能性非常高。对随机选择的克隆进行测试表明它们非常稳定，130 个克隆中只有 4 个在连续生长 80 代后显示出限制性消化片段改变。使用 70 个随机选择的 PAC 进行的 FISH 分析显示没有显着的嵌合克隆。大约 7% 的分析克隆含有与着丝粒区域相关的重复序列，这些区域与一些但不是全部着丝粒杂交。制备DNA板池和超级池，并制备高密度过滤器，每个过滤器包含一式两份的8个板的阵列。对这些超级池和具有 10 个单位点大鼠标记的适当过滤器进行文库筛选，平均发现 8 个阳性克隆，与该文库的估计高基因组覆盖度和大鼠基因组的代表性一致。该库为大鼠基因组分析，特别是多因素疾病模型中涉及的基因的鉴定提供了新的资源。该库和高密度过滤器目前可供科学界使用。
A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614 384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.