Double cos site vectors: simplified cosmid cloning

Abstract

A new vector for construction of cosmid libraries is described. Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two lambda cos sites separated by a blunt-end restriction site. The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms, and the internal blunt-end restriction site prevents cosmid concatemerization. Thus, a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation. The use of this vector system allows efficient cosmid cloning (1 X 10(5) colonies per micrograms insert DNA) and eliminates background due to vector self-ligation. Furthermore, the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse. Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells. This vector contains the Herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence.