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. With decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.
Early attempts at mutagenesis were non-site specific using radiation or chemical mutagens. Analogs of nucleotides and other chemicals were later used to generate localized point mutations, examples of such chemicals are aminopurine, nitrosoguanidine, and bisulfite. Site-directed mutagenesis was achieved in 1973 in the laboratory of Charles Weissmann using a nucleotide analogue N4-hydroxycytidine which induces transition of GC to AT. These methods of mutagenesis however are limited by the kind of mutation they can achieve and they are not as specific as later site-directed mutagenesis methods.
In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with small fragments of phage ϕX174 and restriction nucleases. Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase. For his part in the development of this process, Michael Smith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction.
The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected.
The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture contains both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favors the methylated template DNA, resulting in fewer mutants. Many approaches have since been developed to improve the efficiency of mutagenesis.
Approaches in site-directed mutagenesis
A large number of methods are available to effect site-directed mutagenesis, although most of them are now rarely used in laboratories since the early 2000s as newer techniques allow for simpler and easier way of introducing site-specific mutation into genes.
In 1987 Thomas Kunkel introduced a technique which 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 replicate the phage DNA, its enzymatic machinery may therefore misincorporates dUTP instead of dTTP, resulting in single stranded DNA which 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.
Unlike other methods, cassette mutagenesis need not involve primer extension using DNA polymerase. In this method, a fragment of DNA is synthesized, and then inserted into a plasmid. It involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid. Usually the restriction enzymes that cuts at the plasmid and the oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency, but is limited by the availability of suitable restriction sites flanking the site that is to be mutated.
PCR site-directed mutagenesis
The limitation of restriction sites in cassette mutagenesis may be overcome using polymerase chain reaction with oligonucleotide "primers", such that a larger fragment may be generated covering two convenient restriction sites. The exponential amplification in PCR produces a fragment containing the desired mutation in sufficient quantity to be separate from the original, unmutated plasmid by gel electrophoresis, which may then be inserted in the original context using standard recombinant molecular biology techniques. There are many variations of the same technique. The simplest method places the mutation site towards one of the ends of the fragment whereby one of two oligonucleotides used for generating the fragment contains the mutation. This involves a single step of PCR, but still has the inherent problem of requiring a suitable restriction site near the mutation site unless a very long primer is used. Other variations therefore employ three or four oligionucleotides, two of which may be non-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can be digested and ligated into a plasmid, while the mutagenic oligonucleotide may be complementary to a location within that fragment well away from any convenient restriction site. These methods require multiple steps of PCR so that the final fragment to be ligated can contain the desired mutation.
Whole plasmid mutagenesis
For plasmid manipulations, other site-directed mutagenesis techniques have been largely supplanted by techniques which are highly efficient but relatively simple, easy to use, and commercially available as a kit. An example of these techniques is the Quikchange method, where 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 which is relaxed. 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 which 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-stranded 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 reaction since there is no chain reaction.
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 vivo site-directed mutagenesis methods
- Delitto perfetto
- Transplacement "pop-in pop-out"
- Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker
- Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions
- In vivo site-directed mutagenesis with synthetic oligonucleotides
Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that has improved or special properties.
Investigative tools - specific mutations in DNA allow the function and properties of a DNA sequence or a protein to be investigated in a rational approach.
Commercial applications - proteins may be engineered to produce proteins that are tailored for a specific application. For example, commonly-used laundry detergents may contain subtilisin whose wild-type form has a methionine that can be oxidized by bleach, inactivating the protein in the process. This methionine may be replaced by alanine, thereby making the protein active in the presence of bleach.
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