Mutagenesis (molecular biology technique)
Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, strains of bacteria, or other genetically modified organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also 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 particular cell function to be investigated.
Large number of methods for achieving mutagenesis have been developed. Initially, the kind of mutations artificially induced in laboratory were entirely random, later methods for more specific site-directed mutagenesis were introduced. Since 2013, development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing or mutagenesis of the genome in vivo.
Early approaches to mutagenesis rely on methods which are entirely random in the mutations produced. Cells or organisms may be exposed to mutagens such as UV radiation or mutagenic chemicals, and mutants with desired characteristics are then selected. Hermann Muller discovered that x-rays can cause genetic mutations in fruit flies (published in 1927), and went on to use the Drosophila mutants created for his studies on genetics. For Escherichia coli, mutants may be selected first by exposure to UV radiation, then plated onto agar medium. The colonies formed are then replica-plated, one in rich medium, another in minimal medium, and mutants that have specific nutritional requirements can then be identified by their inability to grow in minimal medium. Similar procedures may be repeated with other types of cells and with different media for selection.
A number of methods for generating random mutations in specific proteins were later developed to screen for mutants with interesting or improved properties. These methods may involve the use of doped nucleotides in oligonucleotides synthesis, or conducting a PCR reaction in conditions that enhance misincorporation of nucleotides (error-prone PCR), for example by reducing the fidelity of replication or using nucleotide analogues. A variation of this method for integrating non-biased mutations in a gene is the Sequence Saturation Mutagenesis. PCR products which contain mutation(s) are then cloned into an expression vector and the mutant proteins produced can then be characterised.
In animal studies, alkylating agents such as N-ethyl-N-nitrosourea (ENU) have been used to generate mutant mice. Ethyl methanesulfonate (EMS) is also often used to generate animal and plant mutants.
In the European union's law (as 2001/18 directive), this kind of mutagenesis produced GMO but the products are exempted from the regulation : no labelling, no evaluation.
Many researchers seek to introduce selected changes to DNA in a site-specific manner. Analogs of nucleotides and other chemicals were first used to generate localized point mutations. Such chemicals include aminopurine, which induces AT to GC transition, while nitrosoguanidine, bisulfite, and N4-hydroxycytidine may induce 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.
Current techniques for site-specific mutation commonly involve using 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 to be introduced at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.
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.
Combinatorial mutagenesis is a technique whereby large number of mutants may be screened for a particular characteristic. In this technique, a few selected positions or a short stretch of DNA may be exhaustively modified to obtain a comprehensive library of mutant proteins. One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic property. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.
In cancer research engineered mutations also provide mechanistic insights into the development of the disease. Insertional mutagenesis using transposons, retrovirus such as mouse mammary tumor virus and murine leukemia virus may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. Various insertional mutagenesis techniques may also be used to study the function of particular gene.
Homologous recombination can be used to produce specific mutation in an organism. Vector containing DNA sequence similar to the gene to be modified is introduced to the cell, and by a process of recombination replaces the target gene in the chromosome. This method can be used to introduce a mutation or knock out a gene, for example as used in the production of knockout mice.
As the cost of DNA oligonucleotides synthesis falls, artificial synthesis of a complete gene is now a viable method for introducing mutations into gene. This method allows for extensive mutation an multiples sites, including the complete redesign of the codon usage of gene to optimise it for a particular organism.
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