Zeng L W, Kreitman M
Department of Ecology and Evolution, University of Chicago, IL 60636, USA.
Biotechniques. 1996 Sep;21(3):446-52. doi: 10.2144/96213st01.
We describe a simple method for constructing subclones containing overlapping nested deletions from cDNA clones (both lambda phage clones and plasmid clones). A PCR-amplified insert is partially digested with 4-cutter restriction enzyme(s). Complete digestion of this DNA with two restriction enzymes, having unique cutting sites at one or the other end of the amplified DNA, creates two sets of overlapping nested subfragments. When recloned into each of two doubly cut pBluescript plasmid vectors, only the two sets of nested subfragments are produced. Minimal nested sets can be constructed by screening subclones using colony PCR, and this set can then be used to determine the entire sequence of the cDNA clone. This method requires only a single cloning step and can be generated from an insert that is amplified directly from a lambda phage clone. This procedure eliminates the sequencing redundancy problem inherent in shotgun cloning, allows large clones to be sequenced using universal primers only and is well-suited for automated DNA-sequencing. Using this method, we successfully sequenced five cDNA clones of five Drosophila subobscura genes.
我们描述了一种简单的方法，用于构建包含来自cDNA克隆（λ噬菌体克隆和质粒克隆）的重叠嵌套缺失的亚克隆。PCR扩增的插入片段用4碱基切割限制酶进行部分消化。用两种在扩增DNA的一端或另一端具有独特切割位点的限制酶对该DNA进行完全消化，产生两组重叠的嵌套亚片段。当重新克隆到两个双酶切的pBluescript质粒载体中时，只会产生两组嵌套亚片段。通过使用菌落PCR筛选亚克隆可以构建最小嵌套集，然后该集合可用于确定cDNA克隆的完整序列。该方法仅需一个克隆步骤，并且可以从直接从λ噬菌体克隆扩增的插入片段生成。该程序消除了鸟枪法克隆中固有的测序冗余问题，允许仅使用通用引物对大克隆进行测序，并且非常适合自动化DNA测序。使用这种方法，我们成功地对五个果蝇亚暗基因的五个cDNA克隆进行了测序。
