Mutagenesis: Site-Directed

ABSTRACT

Site‐directed mutagenesis (SDM) aims to introduce precise alterations in any coding or noncoding deoxyribonucleic acid (DNA) sequence, usually in vitro . These modifications could be as small as a nucleotide or several hundred; in one site or in multisite in the same DNA sequence. Recently, these alterations have been also developed in vivo . SDM success depends on how changes are introduced and mutant selection is done. DNA sequence analysis must be made to verify change(s) before any biochemical or biological experiments are done. Recent methods for SDM and most used commercial kits are discussed. A list of companies offering SDM service is included. The authors have also listed software used for mutagenic oligonucleotide primer‐design. These techniques are revolutionising our understanding of the genetic and molecular mechanisms, protein structure-function relationship, protein-protein interaction and binding sites in any biological system. In addition to the academic benefits of SDM, SDM techniques have impacted biotechnology and the applied field such as engineering new enzymes, drug development, optimisation of heterologous gene expression and secretion. In recent years, technologies employing in vivo gene as well genome modifications (editing) has been significantly improved. CRISPR/Cas9 system for genome editing has already been used in different systems.