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A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322
Authors:M Heusterspreute  J Davison
Institution:Unit of Molecular Biology, International Institute of Cellular and Molecular Pathology, Ac. Hippocrate 75, B-1200 Brussels, Belgium Tel. (02) 762.34.00 Ext: 7463
Abstract:A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.
Keywords:Recombinant DNA  restrictions fragments  analysis of gene function  bp  base pair  tetracycline-sensitive  tetracycline-resistant
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