Sitedirected

Site-directed mutagenesis refers to a set of molecular biology methods used to introduce precise nucleotide changes at a defined location within a DNA sequence. It enables targeted alterations of genes, regulatory elements, or protein-coding regions, allowing researchers to test hypotheses about function or to engineer desired properties.

Most approaches rely on mutagenic primers or templates to propagate a specific change. Early methods used single-stranded DNA templates and mutagenic oligonucleotides; later methods employ polymerase chain reaction (PCR) to generate mutated DNA. Foundational techniques include M13-based mutagenesis, with PCR-based approaches such as overlap extension PCR and megaprimer strategies, and widely used methods like the QuikChange style of mutagenesis.

Site-directed mutagenesis is widely used to study structure–function relationships, to alter enzyme active sites, to improve protein stability or activity, or to create reporter or epitope-tagged constructs. It is commonly applied to plasmid DNA and, with additional strategies, to viral genomes and to some chromosomal targets.

Verification by DNA sequencing is essential, and clone screening is typically required to identify correctly mutated constructs. Limitations include dependence on template quality, potential unintended secondary mutations, and reduced efficiency for larger insertions or multiple changes. In recent years, genome editing technologies such as CRISPR have broadened the scope of site-directed modifications in living cells, often enabling targeted changes directly in genomes, while still relying on the same principle of introducing precise, defined alterations at specific sites.