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Single base, site-directed mutagenesis of a 90 kilobasepair P1 clone

1994; Oxford University Press; Volume: 22; Issue: 20 Linguagem: Inglês

10.1093/nar/22.20.4348

1362-4962

Matthew J. Callow, Lance J. Ferrin, Edward M. Rubin,

Bacterial Genetics and Biotechnology

Site-directed mutagenesis of cloned DNA has been an invaluable tool in the investigation of the regulation and function of genes and their products.However, all approaches are limited by the size of DNA amenable to such procedures and sub-cloning from and into large DNA can be difficult.This report describes a strategy to introduce a point mutation into a 90 kbp P1 plasmid.We utilized RecA Assisted Restriction Endonuclease (RARE) cleavage (1,2) of two restriction sites surrounding the targeted base within the P1 plasmid to permit removal of a small DNA fragment.This fragment was mutated in an M13 vector and re- inserted into the P1, again utilizing RARE cleavage.To simplify screening of potential clones we found it advantageous to include selectable markers during reinsertion that were easily removed,