Zabarovsky E R, Boldog F, Erlandsson R, Kashuba V I, Allikmets R L, Marcsek Z, Kisselev L L, Stanbridge E, Klein G, Sumegi J
Department of Tumor Biology, Karolinska Institutet, Stockholm, Sweden.
Genomics. 1991 Dec;11(4):1030-9. doi: 10.1016/0888-7543(91)90029-e.
A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.
本文描述了一种构建跳跃文库的新方法。该方法的主要特点如下：（1）在文库构建中同时使用两种双相载体（λSK17和λSK22）以提高代表性；（2）采用部分补平反应来消除人工跳跃克隆的克隆，并避免使用选择标记。该方法已用于从EBV基因组甲基化水平较低的淋巴母细胞系CBMI-Ral-STO构建具有代表性的人类NotI跳跃文库（220,000个独立重组克隆）。还构建了来自人3号染色体×小鼠杂交细胞系MCH 903.1的人3号染色体特异性NotI跳跃文库（500,000个独立重组克隆）。在这些重组克隆中，50%-80%代表跳跃到相邻的可切割NotI位点。结合我们先前发表的构建连接文库的方法，该方法使一种新的基因组图谱绘制策略成为可能。该策略包括确定随机连接和跳跃克隆中NotI位点相邻的标签序列。λSK17和λSK22载体的特殊特性便于进行此类测序。获得的STS（序列标签位点）信息可通过计算机组装成代表染色体或整个基因组中NotI位点线性顺序的图谱。计算机化的图谱数据可用于检索感兴趣区域附近的克隆。相应的克隆可从原始克隆库中获得，或者可通过PCR从基因组DNA制备所需的探针。
