Osoegawa K, Woon P Y, Zhao B, Frengen E, Tateno M, Catanese J J, de Jong P J
Department of Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York, 14263, USA.
Genomics. 1998 Aug 15;52(1):1-8. doi: 10.1006/geno.1998.5423.
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.
本文介绍了一些改进方法，这些方法能够构建高度冗余的细菌人工染色体/P1衍生人工染色体文库，插入片段大小更大且相对均匀。载体制备方面的改进以及增强的连接条件减少了背景非重组克隆的数量。固定化高分子量DNA的预电泳去除了克隆过程中的抑制剂，而在单一凝胶内对DNA片段进行两次大小筛选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片段大小。通过电洗脱回收大小分级的DNA片段，而不是更常见的凝胶切片融化及随后的β-琼脂糖酶处理。连接产物的浓缩使得构建所需大小文库所需的电穿孔数量减少了6至12倍。这些改进方法已应用于从人类、小鼠、大鼠、犬类和狒狒基因组制备PAC和BAC文库，平均插入片段大小在160至235kb之间。
