Atchison L, Comis R L, Atchison M L
Fox Chase Center, Philadelphia, PA 19111.
Gene. 1994 Dec 30;151(1-2):325-8. doi: 10.1016/0378-1119(94)90679-3.
We have constructed rare restriction-site (NotI, SacII and ClaI) chromosome 3 (Chr 3)-specific linking libraries in a plasmid-based vector by mass transfer of a lambda phage human Chr-3-specific library (LA03NS01-ATCC57717) into pUC18. Total plasmid DNA isolated from the plasmid-based Chr-3-specific library was digested with either ClaI, NotI or SacII. Linear molecules were separated from undigested circles by pulsed-field polyacrylamide-gel electrophoresis. Purified linear molecules were circularized with T4 DNA ligase and transformed into bacteria. The resulting clones were greatly enriched for sequences recognized by the original restriction endonuclease used for digestion (83 to 95%). These sublibraries are composed of 600 (NotI) 1000 (SacII) or 30,000 (ClaI) clones. Thus, this procedure allows for easy isolation of Chr-3-specific DNA clones containing a variety of rare restriction sites. Sequence-tagged site (STS) data are also presented for five site-specifically mapped Chr-3-specific DNA clones. These studies may facilitate the construction of region specific linking libraries for mapping of various disease-specific loci on Chr 3.
我们通过将λ噬菌体人3号染色体特异性文库（LA03NS01-ATCC57717）大量转移至pUC18中，构建了基于质粒载体的、含有稀有酶切位点（NotI、SacII和ClaI）的3号染色体（Chr 3）特异性连接文库。从基于质粒的Chr-3特异性文库中分离出的总质粒DNA，用ClaI、NotI或SacII进行酶切。通过脉冲场聚丙烯酰胺凝胶电泳将线性分子与未酶切的环状分子分离。纯化后的线性分子用T4 DNA连接酶环化，并转化到细菌中。所得克隆中，被用于消化的原始限制性内切酶识别的序列大量富集（83%至95%）。这些亚文库分别由600个（NotI）、1000个（SacII）或30,000个（ClaI）克隆组成。因此，该方法能够轻松分离出含有各种稀有酶切位点的Chr-3特异性DNA克隆。还给出了5个位点特异性定位的Chr-3特异性DNA克隆的序列标签位点（STS）数据。这些研究可能有助于构建区域特异性连接文库，用于定位3号染色体上各种疾病特异性位点。
