Methods and Concepts in the Life Sciences/Mutagenesis
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. Mutants strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.
Early approaches to mutagenesis rely on methods which are entirely random in the mutations produced. Cells may be exposed to UV radiation or mutagenic chemicals, and mutants with desired characteristic are then selected. For example, Escherichia coli may be exposed to UV radition, 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 requirement can then be identified by its inability to grow in minimal medium and isolated.
A number of methods for generating random mutation in specific protein were later developed to screen for mutants with interesting or improved properties. This may be done by using doped nucleotides in oligonucleotides synthesis, or conducting a PCR reaction in conditions that enhance misincorporation of nucleotide, thereby generating mutants.
In animal studies, N-ethyl-N-nitrosourea (ENU) has been used to generate mutant mice. Ethyl methanesulfonate (EMS) is also often used to generate animal and plant mutants.
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.
Analogs of nucleotides and other chemicals were first used to generate localized point mutations. Such chemicals may be 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 in the kinds of mutants generated nor are they as specific as later methods of site-directed mutagenesis.
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. However, with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.
Whole plasmid mutagenesis
For plasmid manipulations, other site-directed mutagenesis techniques have been supplanted largely by techniques that are highly efficient but relatively simple, easy to use, and commercially available as a kit. An example of these techniques is the Quikchange method, wherein a pair of complementary mutagenic primers are used to amplify the entire plasmid in a thermocycling reaction using a high-fidelity non-strand-displacing DNA polymerase such as pfu polymerase. The reaction generates a nicked, circular DNA. The template DNA must be eliminated by enzymatic digestion with a restriction enzyme such as DpnI, which is specific for methylated DNA. All DNA produced from most Escherichia coli strains would be methylated; the template plasmid that is biosynthesized in E. coli will, therefore, be digested, while the mutated plasmid, which is generated in vitro and is therefore unmethylated, would be left undigested. Note that, in these double-strand plasmid mutagenesis methods, while the thermocycling reaction may be used, the DNA need not be exponentially amplified as in a PCR. Instead, the amplification is linear, and it is therefore inaccurate to describe them as a PCR, since there is no chain reaction.
Note that pfu polymerase can become strand-displacing at higher extension temperature (≥70 °C) which can result in the failure of the experiment, therefore the extension reaction should be performed at the recommended temperature of 68 °C. In some applications, this method has been observed to lead to insertion of multiple copies of primers. A variation of this method, called SPRINP, prevents this artifact and has been used in different types of site directed mutagenesis.
In 1987, Thomas Kunkel introduced a technique that reduces the need to select for the mutants. The DNA fragment to be mutated is inserted into a phagemid such as M13mp18/19 and is then transformed into an E. coli strain deficient in two enzymes, dUTPase (dut) and uracil deglycosidase (ung). Both enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly synthesized DNA. As the double-mutant E. coli replicates the phage DNA, its enzymatic machinery may, therefore, misincorporate dUTP instead of dTTP, resulting in single-strand DNA that contains some uracils (ssUDNA). The ssUDNA is extracted from the bacteriophage that is released into the medium, and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heteroduplex DNA that forms consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP. The DNA is then transformed into an E. coli strain carrying the wildtype dut and ung genes. Here, the uracil-containing parental DNA strand is degraded, so that nearly all of the resulting DNA consists of the mutated strand.