DIRECTIONAL CLONING OF DNA FRAGMENTS AT A LARGE DISTANCE FROM AN INITIAL PROBE - A CIRCULARIZATION METHOD
DIRECTIONAL CLONING OF DNA FRAGMENTS AT A LARGE DISTANCE FROM AN INITIAL PROBE - A CIRCULARIZATION METHOD
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DOI：
10.1073/pnas.81.21.6812
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
WEISSMAN, SM
中科院分区：
文献类型：
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作者：
COLLINS, FS;WEISSMAN, SM
介绍了一种[人类]DNA克隆程序的原理，该程序可以在距离初始探针5-2000千碱基的地方定向生成基因组DNA片段。该方法依赖于高分子量基因组DNA的部分消化和随后的结扎在非常低的浓度，以产生共价DNA环。然后可以构建这些圆的连接片段库。通过将标记DNA片段合并到共价圈中，可以实现结块的生物或物理选择。一个45千碱基的宇宙碎片已被成功地用于测试该程序。在适当低的结扎浓度下(0.8 .mu。.simeq g / ml)。90%的结扎DNA以单体环状存在。较大的DNA片段将需要降低DNA浓度作为DNA长度的倒数平方根。选择tRNA抑制基因作为选择性标记基因。将消化的圆圈结扎成琥珀突变的。lambda。噬菌体在宿主中的繁殖只允许含有连接片段的噬菌体产生斑块。提出并讨论了这种方法的潜在应用，例如复杂遗传位点的定位或从链接基因向感兴趣的基因移动。
The principle of a [human] DNA cloning procedure that directionally generates genomic DNA fragments 5-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high MW genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 .mu.g/ml), .simeq. 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene was tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated .lambda. phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.