Practice question

Site-directed mutagenesis introduces defined nucleotide changes at predetermined position to assess functional impact on protein or regulatory element. Methodology employs synthetic oligonucleotide primers that are complementary to target plasmid except for desired substitution, insertion, or deletion in central region. These mismatched primers anneal during polymerase chain reaction amplification, incorporating mutation into newly synthesized strands. Resulting plasmids carry mutation and are selected after digestion of methylated parental template. Random mutagenesis creates unpredictable changes, while EMSA and footprinting assay DNA binding, not generate mutations. Therefore primer-directed approach provides precision for structure-function analysis.