Multipoint Mutagenesis for the Generation of Enzyme Libraries

Abstract

A one step PCR mutagenesis method has been tested for generating a focused mutant library. Seven positions have been targeted simultaneously by using a mixture of double stranded mutagenic oligonucleotides, PCR, DpnI digestion and transformation into an expression host for screening. Library sizes of several thousands of mutants could easily be achieved. Sequencing of randomly picked clones revealed a regular distribution of the number of introduced mutations. No wildtype sequence was found, but “hot spots” that do not closely correlate with the melting temperature, the GC percentage or the length of the primers have been observed.