Kai T, Song J C, Tashiro H, Saka F, Ozawa K, Miwa T, Kitabayashi I, Soeda E, Yokoyama K
Gene Bank, RIKEN (Institute of Physical and Chemical Research), Ibaraki, Japan.
FEBS Lett. 1990 Nov 26;275(1-2):77-82. doi: 10.1016/0014-5793(90)81443-r.
A simple method for the molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector (in situ YAC construction) [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment-YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.
先前报道了一种通过将细胞包裹在琼脂糖珠中来分子克隆超过一百千碱基对外源DNA片段的简单方法，用于在酵母人工染色体（YAC）载体中构建人类基因组DNA文库（原位YAC构建）[1]。该方法的效率在含有人类DNA片段的琼脂糖珠在转化前熔化的步骤中受到损害。连接的人类DNA片段-YAC载体的不完全溶解性常常导致低于理想的转化频率。为克服这个问题，我们开发了一种新的改进方法，该方法涉及使用琼脂糖膜。本文讨论了构建人类DNA大片段克隆所涉及的技术操作。
