page_title: Mutagenesis (molecular biology technique)
section_title: Site-directed mutagenesis
hierarchical_section_title: Mutagenesis (molecular biology technique) / Site-directed mutagenesis
caption_attribution_description: English: Site Directed Mutagenesis
context_page_description: In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.
  Many methods of mutagenesis exist today. Initially, the kind of mutations artificially induced in the laboratory were entirely random; however, with the development of site-directed mutagenesis, more specific changes can be made. Since 2013, development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has allowed for the editing or mutagenesis of a genome in vivo. Site directed mutagenesis has proved useful in situations that random mutagenesis is not.
context_section_description: Random mutagenesis techniques provide an advantage in terms of control of how many mutations are added. UV mutagenesis allows for the change in single nucleotides, however it does not offer much control as to which nucleotide is being changed. Many researchers seek to introduce selected changes to DNA in a precise, site-specific manner. Prior to site directed mutation, all mutations made were random, following mutation scientists had to use selection for the desired phenotype to attain the desired mutation. Many researchers seek to introduce selected changes to DNA in a precise, site-specific manner. Early attempts uses analogs of nucleotides and other chemicals were first used to generate localized point mutations. Such chemicals include aminopurine, which induces an AT to GC transition, while nitrosoguanidine, bisulfite, and N⁴-hydroxycytidine may induce a GC to AT transition. These techniques allow specific mutations to be engineered into a protein; however, they are not flexible with respect to the kinds of mutants generated, nor are they as specific as later methods of site-directed mutagenesis and therefore have some degree of randomness. Other technologies such as cleavage of DNA at specific sites on the chromosome, addition of new nucleotides, and exchanging of base pairs it is now possible to decide where mutations can go.
  Current techniques for site-specific mutation commonly involve using pre-fabricated mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation or deletion or insertion of small stretches of DNA at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.
  Newer and more efficient methods of site directed mutagenesis are being constantly developed. A new technique has been developed called "Seamless ligation cloning extract" (or SLiCE for short) that allows for the cloning of certain sequences of DNA within the genome. This method is now used in Polymerase chain reactions to make site directed mutagenesis more efficient by allowing more than one DNA fragment to be inserted into the genome at once.
  The uses for site directed mutagenesis are seemingly endless, however one important function is what the mutations tell us once they have occurred. Site directed mutations were recently used to determine how susceptible certain species were to chemicals that are often used In labs. The experiment used site directed mutagenesis to mimic the expected mutations of the specific chemical. The mutation resulted in a change in specific amino acids and the affects of this mutation were analyzed.
  The site-directed approach may be done systematically in such techniques as alanine scanning mutagenesis, whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein. Another comprehensive approach is site saturation mutagenesis where one codon or a set of codons may be substituted with all possible amino acids at the specific positions.
caption: Simplified diagram of the site directed mutagenic technique using pre-fabricated oligonucleotides in a primer extension reaction with DNA polymerase
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