Phage Lambda and Molecular Cloning

Abstract

For more than 20 years, transducing derivatives of lambdoid phages have facilitated functional and structural analyses of bacterial genes. Restriction enzymes that make staggered cuts within specific DNA sequences (targets) produce discrete fragments with short cohesive ends (Hedgpeth et al. 1972; Mertz and Davis 1972; Bigger et al. 1973), and this discovery immediately indicated that fragments of DNA could be spliced into the severed arms of a λ vector molecule. The cohered fragments would then be joined covalently by DNA ligase (Gellert 1967), and the recombinant genomes, transducing phages, recovered by transfection of Escherichia coli (Mandel and Higa 1970). This goal was quickly realized (Murray and Murray 1974; Thomas et al. 1974), and detailed analyses of the type previously possible for only a few E. coli genes became generally applicable. The ability to insert DNA fragments from any source into plasmid and phage vectors, supplemented with the new techniques for rapid DNA sequence determination (Maxam and Gilbert 1977; Sanger et al. 1977), has already generated many exciting advances in diverse areas of molecular biology.

The development of the phage λ chromosome as a receptor for fragments of DNA was assured by the availability of well-characterized deletion mutants, by the means of selecting for the loss of restriction targets and by prior identification of the essential components of the phage transcriptional circuits and replication system. The precise knowledge attained by 1972 showed the genetic engineer which regions of the λ genome could be sacrificed and, also, which special features could be...