7 - Cosmid Cloning with Small Genomes

Abstract

This chapter discusses various aspects of cosmid cloning with small genomes. Cosmids are defined as plasmids containing one or more cohesive sites of bacteriophage X DNA. After linearization of cosmids by digestion with one or more restriction enzymes and ligation to 40–50-kbp DNA, among the ligation products are linear polymer DNA molecules containing cos sites spaced between 37 and 51 kbp apart. A specially designed multiple cloning site (MCS) contains recognition sequences for the restriction endonucleases used for cloning flanked by recognition sequences for endonucleases. Endonucleases that recognize 6-bp restriction sites are more convenient for restriction mapping. Enzymes recognizing 4-bp restriction sites ensure a better random fragmentation. Establishing contigs can be accomplished through the use of both landmarking and walking procedures, carried out in parallel. Landmarking analysis should be performed with candidates believed to overlap among the clone selected for restriction analysis. Chromosome walking analysis should be initiated with 10–20 or more clones without apparent overlap. It is suggested that a genomic library permits construction of a high-resolution physical map which can be converted to a detailed genetic map by identifying and localizing genes of interest on the physical map.