An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA. This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci. Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process. First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s). Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants. Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods. The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system. Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs. Very high efficiency of mutation incorporation and selection were obtained in all cases. Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants. The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)