Studies on oligonucleotide-directed mutagenesis of Escherichia coli 5S rRNA

The plasmid pKK5-l was purified from Escherichia coli strain HB101 and digested with HindIII. The restriction fragment containing the 5S rDNA was isolated by agarose gel electrophoresis and cloned into the HindIII site of the single-stranded phage vector M13 mp18. The recombinants produced white plaques on IPTG / XGal plates. Single-stranded DNA was isolated from several white plaques and the orientation of inert was determined by sequencing with a M13 universal primer. A 17-base mutagenic oligonucleotide was synthesized by manual phosphoramidite method and site- directed mutagenesis of the 5S rRNA was attempted by a two primer method. All of the individual procedures required to conduct site-directed mutagenesis were successfully implemented. Unfortunately no mutants were obtained. Possible explanations for the failure to recover the desired mutant and some recommendations for overcoming the apparent problems are discussed.