An improved approach for construction of bacterial artificial chromosome libraries
An improved approach for construction of bacterial artificial chromosome libraries
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DOI：
10.1006/geno.1998.5423
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发表时间：
1998-08-15
期刊：
影响因子：
4.4
通讯作者：
de Jong, PJ
中科院分区：
文献类型：
--
作者：
Osoegawa, K;Woon, PY;de Jong, PJ
本文提出了改进的方法，其使得能够产生高度冗余的细菌人工染色体/P1衍生的人工染色体文库，其具有更大且相对均匀的插入片段大小。载体制备的改进和增强的连接条件减少了背景非重组克隆的数量。固定的高分子量DNA的预电泳去除了克隆过程的抑制剂，而在单个凝胶内对DNA片段进行两次大小测定有效地消除了小的限制性片段，从而增加了克隆的平均插入大小。通过电洗脱而不是随后用β-琼脂酶处理的更常见的凝胶切片的熔化来回收大小分级的DNA片段，连接产物的浓度使制备所需大小的文库所需的电穿孔次数减少6至12倍。这些改进的方法已被应用于从人、鼠、大鼠、犬和狒狒基因组制备PAC和BAC文库，平均插入片段大小在160和235 kb之间。(C)北京：科学出版社.
Presented here are improved methodologies that enable the generation of highly redundant bacterial artificial chromosome/P1-derived artificial chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones, The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent beta-agarase treatment, Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb. (C) 1998 Academic Press.