11] Construction and use of pBR322 plasmids that yield single-stranded DNA for sequencing

Abstract

This chapter discusses the construction and use of pBR322 plasmids that yield single-stranded deoxyribonucleic acid (DNA) for sequencing. The chapter describes a set of chimeric cloning vectors with regard to the yield of single-stranded DNA produced. The chapter also describes a procedure for the construction of chimeric plasmids in detail. The filamentous male-specific coliphages, Ff (fl, M13, fd), only infect Escherichia coli cells that contain an F pilus and are one of the smallest known single-stranded DNA bacteriophages. DNA replication of the phage involves three principal steps. In the first step, synthesis of the complementary strand converts the single-stranded viral DNA into a double-stranded replicative form (RF). Viral or (+)-strand DNA replication in the second step is initiated by the introduction of a specific nick on the (+) strand of the RF molecule by viral gene II protein. In the third step, the concentration of viral gene V protein, a single-stranded DNA binding protein, reaches a level high enough to coat the newly replicated single-stranded (+) DNA. The yield of single-stranded plasmid DNA varies with the type of helper phage used in the infection. Some of the mutant helper phages have mutations within gene II that alter either the level or activity of gene II protein.