Hultman T, Uhlén M
Royal Institute of Technology, Department of Biochemistry, Stockholm, Sweden.
J Biotechnol. 1994 Jun 30;35(2-3):229-38. doi: 10.1016/0168-1656(94)90038-8.
A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication. After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation. These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences. Thus, cloning can be achieved without the use of ligase or restriction enzymes. The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.
本文描述了一种从λ噬菌体或黏粒文库中创建适用于DNA测序的亚克隆的固相方法。将纯化的目标DNA进行超声处理，并将两个接头（其中一个寡核苷酸在5'端进行了生物素化）连接到超声处理产生的片段末端。经过大小分离后，将片段固定在固相支持物上，并洗脱每个固定片段的非生物素化链。通过这种方式，获得了一个单链片段文库。所有片段都包含由接头连接引入的22个碱基的“通用”侧翼序列。这些侧翼序列随后可用于固相克隆到含有互补序列的单链载体中。因此，无需使用连接酶或限制酶即可实现克隆。所得的亚克隆用于直接固相测序，固定的链可用于从单链片段文库中选择性去除同源DNA。因此，可以创建一个未测序片段的亚文库。在这里，我们表明从λ噬菌体DNA开始可以获得适用于直接固相测序的克隆文库。研究了同源和非同源片段的选择性杂交效率。讨论了将这种方法用于大规模基因组和cDNA测序的自动化克隆策略的可能性。
