Site-specific mutagenesis restriction—selection

Restriction enzyme-mediated integration (REMI) is a commonly used method for random mutagenesis that has been adapted for the use in D. discoideum by Kuspa and Loomis [40]. A plasmid containing an appropriate selection marker is Hn-earized with a restriction enzyme, and the linear plasmid is transformed by electroporation along with the restriction enzyme (Fig. 5.2). It is assumed that the restriction enzyme will cut certain chromosomal loci at specific recognition sites. If a transformed plasmid would Hgate with its sticky ends to the double-strand break introduced by the restriction enzyme, mutants can be isolated based on the selection marker present on the integrated REMI plasmid. 

It has been shown that cis - DDP, at low platinum/nucleotide ratios selectively inhibits the activity of several restriction endo - and exo nucleases when their cutting sites are adjacent to (d6)n (d )n sequences with n > 2 This demonstrates the sequence specificity of the binding of cis - DDP to DNA (17, 18, 19). GAG and GCG sequences also appear to be selectively involved in base - pair substitution mutagenesis and this suggests the possibility of platinum chelation by two guanines separated by a third base (20). 

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