Orbach M J
Dupont Experimental Station, Wilmington, DE 19880-0402.
Gene. 1994 Dec 2;150(1):159-62. doi: 10.1016/0378-1119(94)90877-x.
The construction of a double-cos-site cosmid vector, pMOcosX, for use in making filamentous fungal genomic DNA libraries, is described. The vector has features that allow for selection of clones introduced into fungi by transformation and for efficient chromosome walking experiments. These features include (i) two cos sites allowing for easy construction of libraries without requiring size selection of insert DNA; (ii) an XhoI site for insertion of Sau3AI or MboI partially digested genomic DNA inserts that allows usage of a half-site fill-in method which minimizes the possibility of producing clones containing chimeric inserts; (iii) a bacterial hygromycin phosphotransferase-encoding gene fused to a modified cpc-1 promoter of Neurospora crassa for direct selection of cosmid clones upon introduction into fungal cells; and (iv) T7 and T3 bacteriophage promoters and EcoRI, NotI and BamHI restriction sites flanking the cloning site that allow for synthesis of, or isolation of, end-specific probes for chromosome walking. The combination of features in this vector allows for the easy construction and use of high-quality fungal DNA libraries from small amounts of genomic DNA.
本文描述了一种用于构建丝状真菌基因组DNA文库的双粘性末端粘粒载体pMOcosX的构建方法。该载体具有一些特性，可用于筛选通过转化导入真菌的克隆，并进行高效的染色体步移实验。这些特性包括：（i）两个粘性末端位点，无需对插入DNA进行大小选择即可轻松构建文库；（ii）一个XhoI位点，用于插入Sau3AI或MboI部分消化的基因组DNA插入片段，允许使用半位点填充方法，最大限度地减少产生含有嵌合插入片段克隆的可能性；（iii）一个与粗糙脉孢菌修饰的cpc-1启动子融合的细菌潮霉素磷酸转移酶编码基因，用于在将粘粒克隆导入真菌细胞后直接筛选；（iv）位于克隆位点两侧的T7和T3噬菌体启动子以及EcoRI、NotI和BamHI限制性酶切位点，可用于合成或分离用于染色体步移的末端特异性探针。该载体的这些特性组合使得能够从少量基因组DNA轻松构建和使用高质量的真菌DNA文库。
