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 one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. However, with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.

History

Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific, generating random mutations.[1] Analogs of nucleotides and other chemicals were later used to generate localized point mutations,[2] examples of such chemicals are aminopurine,[3] nitrosoguanidine,[4] and bisulfite.[5] 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.[6][7] 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.[8][9] 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.[10] 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.

Basic mechanism

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.

Approaches

A large number of methods are available to effect site-directed mutagenesis,[11] 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.

Kunkel's method

In 1985, Thomas Kunkel introduced a technique that reduces the need to select for the mutants.[12] 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.

Cassette mutagenesis

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.[13] 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.

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 separated 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 toward 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 oligonucleotides, 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, whereas 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. The design process for generating a fragment with the desired mutation and relevant restriction sites can be cumbersome. Software tools like SDM-Assist[14] can simplify the process.

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,[15] 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.[16] A variation of this method, called SPRINP, prevents this artifact and has been used in different types of site directed mutagenesis.[16]

In vivo site-directed mutagenesis methods

Applications

Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that has improved or special properties(i.e.protein engineering).

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 mutant forms 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, significantly reducing the activity the protein in the process.[19] This methionine may be replaced by alanine or other residues, making it resistant to oxidation thereby keeping the protein active in the presence of bleach.[20]

Gene synthesis

As the cost of DNA oligonucleotides synthesis falls, artificial synthesis of a complete gene is now a viable method for introducing mutation into gene. This method allows for extensive mutagenesis over multiples sites, including the complete redesign of the codon usage of gene to optimise it for a particular organism.[21]

See also

References

  1. ↑ Kilbey, B. J. (1995). "Charlotte Auerbach (1899-1994)". Genetics. 141 (1): 1–5. PMC 1206709Freely accessible. PMID 8536959.
  2. ↑ Shortle, D.; Dimaio, D.; Nathans, D. (1981). "Directed Mutagenesis". Annual Review of Genetics. 15: 265–294. doi:10.1146/annurev.ge.15.120181.001405. PMID 6279018.
  3. ↑ Caras, I. W.; MacInnes, M. A.; Persing, D. H.; Coffino, P.; Martin Jr, D. W. (1982). "Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells". Molecular and Cellular Biology. 2 (9): 1096–1103. PMC 369902Freely accessible. PMID 6983647.
  4. ↑ McHugh, G. L.; Miller, C. G. (1974). "Isolation and Characterization of Proline Peptidase Mutants of Salmonella typhimurium". Journal of Bacteriology. 120 (1): 364–371. PMC 245771Freely accessible. PMID 4607625.
  5. ↑ D Shortle & D Nathans (1978). "Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome.". Proceedings of the National Academy of Sciences. 75 (5): 2170–2174. doi:10.1073/pnas.75.5.2170. PMC 392513Freely accessible. PMID 209457.
  6. ↑ R A Flavell; D L Sabo; E F Bandle & C Weissmann (1975). "Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA". Proc Natl Acad Sci U S A. 72 (1): 367–371. doi:10.1073/pnas.72.1.367. PMC 432306Freely accessible. PMID 47176.
  7. ↑ Willi Müller; Hans Weber; François Meyer; Charles Weissmann (1978). "Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123". Journal of Molecular Biology. 124 (2): 343–358. doi:10.1016/0022-2836(78)90303-0. PMID 712841.
  8. ↑ Hutchison Ca, 3.; Edgell, M. H. (1971). "Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid". Journal of Virology. 8 (2): 181–189. PMC 356229Freely accessible. PMID 4940243.
  9. ↑ Marshall H. Edgell, Clyde A. Hutchison, III, and Morton Sclair (1972). "Specific Endonuclease R Fragments of Bacteriophage X174 Deoxyribonucleic Acid". Journal of Virology. 9 (4): 574–582. PMC 356341Freely accessible. PMID 4553678.
  10. ↑ Hutchison CA, Phillips S, Edgell MH, Gillam S, Jahnke P, Smith M (September 1978). "Mutagenesis at a specific position in a DNA sequence" (PDF). J. Biol. Chem. 253 (18): 6551–60. PMID 681366.
  11. ↑ Braman, Jeff, ed. (2002). In Vitro Mutagenesis Protocols. Methods in Molecular Biology. 182 (2nd ed.). Humana Press. ISBN 978-0896039100.
  12. ↑ Kunkel TA. (1985). "Rapid and efficient site-specific mutagenesis without phenotypic selection." (PDF). Proceedings of the National Academy of Sciences. 82 (2): 488–92. doi:10.1073/pnas.82.2.488. PMC 397064Freely accessible. PMID 3881765.
  13. ↑ Wells, J. A.; Estell, D. A. (1988). "Subtilisin--an enzyme designed to be engineered". Trends in Biochemical Sciences. 13 (8): 291–297. doi:10.1016/0968-0004(88)90121-1. PMID 3154281.
  14. ↑ Karnik, Abhijit; Karnik, Rucha; Grefen, Christopher (2013). "SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites". BMC Bioinformatics. 14 (1): 105. doi:10.1186/1471-2105-14-105. ISSN 1471-2105.
  15. ↑ Papworth, C., Bauer, J. C., Braman, J. and Wright, D. A. (1996). "Site-directed mutagenesis in one day with >80% efficiency.". Strategies. 9 (3): 3–4.
  16. 1 2 Edelheit, O; Hanukoglu, A; Hanukoglu, I (2009). "Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies". BMC Biotechnol. 9: 61. doi:10.1186/1472-6750-9-61. PMC 2711942Freely accessible. PMID 19566935.
  17. ↑ Storici F.; Resnick MA. (2006). "The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.". Methods in Enzymology. 409: 329–45. doi:10.1016/S0076-6879(05)09019-1. PMID 16793410.
  18. ↑ Storici F.; Resnick MA (2003). "Delitto perfetto targeted mutagenesis in yeast with oligonucleotides". Genetic Engineering. 25: 189–207. PMID 15260239.
  19. ↑ Stauffer CE, Etson D (October 10, 1969). "The effect on subtilisin activity of oxidizing a methionine residue". Journal of Biological Chemistry. 244 (19): 5333–8. PMID 5344139.
  20. ↑ Estell DA, Graycar TP, Wells JA (10 June 1985). "Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation". Journal of Biological Chemistry. 260 (11): 6518–21. PMID 3922976.
  21. ↑ Yury E. Khudyakov, Howard A. Fields, ed. (25 September 2002). Artificial DNA: Methods and Applications. CRC Press. p. 13. ISBN 9781420040166.

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