[1] Production of single-stranded plasmid DNA

Publisher Summary This chapter discusses the production of single-stranded plasmid DNA. The chapter focuses on M13KO7 and its uses. The chapter reviews M13 biology; M13 mutants play a vital role in the functioning of M13KO7. M13 is a phage that contains a circular single-stranded DNA (ssDNA) molecule of 6407 bases packaged in a filamentous virion, which is extruded from the cell without lysis. It can infect only cells having F-pili to which it binds for entering the cell. The phage genome consists of nine genes encoding 10 proteins and contains an intergenic region of 508 bases. Phage replication consists of three phases: (1) ss-double strand (ds), (2) ds-ds, and (3) ds-ss. The intergenic region (IG) structure contains regions important for four phage processes: (1) the sequences necessary for the recognition of an ssDNA by phage proteins for its efficient packaging into viral particles; (2) the site of synthesis of an RNA primer that is used to initiate strand synthesis; (3) the initiation; and (4) the termination of (+) strand synthesis. In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated. The origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.