Site-directed mutagenesis

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. 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. Site-directed mutagenesis is one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. There are numerous methods for achieving site-directed mutagenesis, but with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis. Since 2013, the development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing of the genome, and mutagenesis may be performed in vivo with relative ease. Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific, generating random mutations. 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 1974 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-strand 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 by DNA sequencing to check that they contain the desired mutation. The original method using single-primer extension was inefficient due to a low yield of mutants. This resulting mixture contains both the original unmutated template as well as the mutant strand, producing a mixed population of mutant and non-mutant progenies. Furthermore, the template used is methylated while the mutant strand is unmethylated, and the mutants may be counter-selected due to presence of mismatch repair system that favors the methylated template DNA, resulting in fewer mutants. Many approaches have since been developed to improve the efficiency of 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 ways of introducing site-specific mutation into genes. In 1985, 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 (udg). 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 udg 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 cut 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.