WordNet

straightforward in means or manner or behavior or language or action; "a direct question"; "a direct response"; "a direct approach"
having no intervening persons, agents, conditions; "in direct sunlight"; "in direct contact with the voters"; "direct exposure to the disease"; "a direct link"; "the direct cause of the accident"; "direct vote" (同)unmediated
in precisely the same words used by a writer or speaker; "a direct quotation"; "repeated their dialog verbatim" (同)verbatim
(of a current) flowing in one direction only; "direct current"
be in charge of
being an immediate result or consequence; "a direct result of the accident"
command with authority; "He directed the children to do their homework"
direct in spatial dimensions; proceeding without deviation or interruption; straight and short; "a direct route"; "a direct flight"; "a direct hit"
give directions to; point somebody into a certain direction; "I directed them towards the town hall"
guide the actors in (plays and films)
lacking compromising or mitigating elements; exact; "the direct opposite"
moving from west to east on the celestial sphere; or--for planets--around the sun in the same direction as the Earth
similar in nature or effect or relation to another quantity; "a term is in direct proportion to another term if it increases (or decreases) as the other increases (or decreases)"
serve in a specific professional capacity; "the priest sat for confession"; "she sat on the jury"
be around, often idly or without specific purpose; "The object sat in the corner"; "We sat around chatting for another hour" (同)sit around
be seated (同)sit_down
be in session; "When does the court of law sit?"
be located or situated somewhere; "The White House sits on Pennsylvania Avenue"
the piece of land on which something is located (or is to be located); "a good site for the school" (同)land site
physical position in relation to the surroundings; "the sites are determined by highly specific sequences of nucleotides" (同)situation
manageable by a supervising agent; "a directed program of study"
(often used in combination) having a specified direction; "a positively directed vector"; "goal-directed"
an event capable of causing a mutation

PrepTutorEJDIC

『まっすぐな』,一直線の(straight),直進する / (列車などが途中駅に寄らずに)直行 / 直系の / 『直接の』,じかの / 『率直な』,単刀直入の / 全くの,絶対の / 原典どおりの / 『まっすぐに』,直行で / 〈事〉‘を'『指揮する』,監督する / 《文》〈人〉‘に'『命令する』,指図する / 〈人〉‘に'(…への)『道を教える』《+『名』〈人〉+『to』+『名』》 / (…に)〈目・注意・努力など〉‘を'『向ける』《+『名』+『to』(『at, toward』)+『名』(do『ing』)》 / 〈音楽・演劇・映画など〉‘を'『指揮する』,演出する,監督する / (…に)〈手紙など〉‘を'あてる《+『名』+『to』+『名』》
(…に)『座る』,座っている《+『at』(『on, in』)+『名』》・着席する《+『down』》・(…に)〈鳥などが〉『止まる』,休む《+『on』+『名』》・〈めんどりが〉卵を抱く,巣に就く / 《場所の副詞[句]を伴って》(ある場所に)『位置する』・ (画家・写真家のために)ポーズをとる・しっとしている,動かないでいる・(議員・委員などの)職に就いている《+『on』+『名』》・〈議会・法廷などが〉開会(開廷)される・(…に)負担となる,重荷となる《+『on』(『upon』)+『名』》・〈衣服などが〉(…に)合う,似合う《+『on』+『名』》・〈人〉‘を'座らせる,着席させる《+『down』+『名,』+『名』+『down』》・〈馬〉‘に'乗る・《英》〈試験〉‘を'受ける
(町・建物などの)『場所』;敷地,用地 / 遺跡;(事件などの)現場 / 〈建物など〉‘を'位置させる(locate)

Wikipedia preview

出典(authority):フリー百科事典『ウィキペディア（Wikipedia）』「2017/03/28 16:04:20」(JST)

wiki en

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. Since 2013, 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.^[1]

History

Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific, generating random mutations.^[2] Analogs of nucleotides and other chemicals were later used to generate localized point mutations,^[3] examples of such chemicals are aminopurine,^[4] nitrosoguanidine,^[5] and bisulfite.^[6] 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.^[7]^[8] 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.^[9]^[10] 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.^[11] 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,^[12] 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.^[13] 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.^[14] 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^[15] 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,^[16] 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.^[17] A variation of this method, called SPRINP, prevents this artifact and has been used in different types of site directed mutagenesis.^[17]

In vivo site-directed mutagenesis methods

Delitto perfetto^[18]
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^[19]

Applications

Site-directed mutagenesis is used to generate mutations that may produce a 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.^[20] 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.^[21]

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.^[22]

References

^ Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPR-Cas9 for genome engineering". Cell. 157 (6): 1262–78. doi:10.1016/j.cell.2014.05.010. PMC 4343198. PMID 24906146.
^ Kilbey, B. J. (1995). "Charlotte Auerbach (1899-1994)". Genetics. 141 (1): 1–5. PMC 1206709. PMID 8536959.
^ 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.
^ 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 369902. PMID 6983647.
^ 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 245771. PMID 4607625.
^ 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 392513. PMID 209457.
^ 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 432306. PMID 47176.
^ 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.
^ 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 356229. PMID 4940243.
^ 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 356341. PMID 4553678.
^ 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.
^ Braman, Jeff, ed. (2002). In Vitro Mutagenesis Protocols. Methods in Molecular Biology. 182 (2nd ed.). Humana Press. ISBN 978-0896039100.
^ 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 397064. PMID 3881765.
^ 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.
^ 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.
^ 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.
^ ^a ^b 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 2711942. PMID 19566935.
^ 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.
^ Storici F.; Resnick MA (2003). "Delitto perfetto targeted mutagenesis in yeast with oligonucleotides". Genetic Engineering. 25: 189–207. PMID 15260239.
^ 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.
^ 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.
^ Yury E. Khudyakov, Howard A. Fields, ed. (25 September 2002). Artificial DNA: Methods and Applications. CRC Press. p. 13. ISBN 9781420040166.

External links

Nobel Lecture on Invention of Site-Directed Mutagenesis
OpenWetWare
Diagram summarizing site-directed mutagenesis

UpToDate Contents

全文を閲覧するには購読必要です。 To read the full text you will need to subscribe.

1. 原発不明の低分化癌 poorly differentiated carcinoma of unknown primary site
2. 原発不明の低分化腫瘍 poorly differentiated neoplasms of unknown primary site
3. 原発不明の腺癌 adenocarcinoma of unknown primary site
4. 持続的腹膜透析におけるトンネルおよび腹膜カテーテル出口部位の感染 tunnel and peritoneal catheter exit site infections in continuous peritoneal dialysis
5. 常染色体優性多発性嚢胞腎の遺伝学および嚢胞成長のメカニズム genetics of autosomal dominant polycystic kidney disease and mechanisms of cyst growth

English Journal

Improvement of hydrogen peroxide stability of Pleurotus eryngii versatile ligninolytic peroxidase by rational protein engineering.

Bao X, Huang X, Lu X, Li JJ.SourceKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Enzyme and microbial technology.Enzyme Microb Technol.2014 Jan 10;54:51-8. doi: 10.1016/j.enzmictec.2013.10.003. Epub 2013 Oct 16.
Peroxide tolerant versatile peroxidases are required for industrial applications. In this study, rational protein engineering was performed to improve the oxidative stability of Pleurotus eryngii versatile ligninolytic peroxidase. Residues which are easily oxidized such as methionine, and close to H
PMID 24267568

The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris.

Han M, Wang X, Ding H, Jin M, Yu L, Wang J, Yu X.SourceKey Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, China; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
Enzyme and microbial technology.Enzyme Microb Technol.2014 Jan 10;54:32-7. doi: 10.1016/j.enzmictec.2013.09.014. Epub 2013 Oct 8.
The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia
PMID 24267565

The epitope structure of Citrus tristeza virus coat protein mapped by recombinant proteins and monoclonal antibodies.

Wu GW, Tang M, Wang GP, Wang CX, Liu Y, Yang F, Hong N.SourceNational Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China.
Virology.Virology.2014 Jan 5;448C:238-246. doi: 10.1016/j.virol.2013.10.021. Epub 2013 Oct 31.
It has been known that there exists serological differentiation among Citrus tristeza virus (CTV) isolates. The present study reports three linear epitopes (aa 48-63, 97-104, and 114-125) identified by using bacterially expressed truncated coat proteins and ten monoclonal antibodies against the nati
PMID 24314654

Japanese Journal

A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

Sato-Masumoto Naoko,Ito Michiho
Phytochemistry 102, 46-54, 2014-06
… In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. …
NAID 120005439487

Evolutionary analysis of the functional plasticity of Staphylococcus aureus C₃₀ carotenoid synthase

Furubayashi Maiko,Saito Kyoichi,Umeno Daisuke
Journal of bioscience and bioengineering 117(4), 431-436, 2014-04
… In this study, we performed a directed evolution of the C_<40>-specialist variant CrtM_<F26A> … Examination of the resultant mutants, together with the site-directed mutagenesis study identified three new mutations (H12A, D27A and 1240F) that affect the size specificity of this enzyme. …
NAID 110009817057

Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase

Briggs Gabrielle D.,Bulley Jesse,Dickson Phillip W.
The journal of biochemistry 155(3), 183-193, 2014-03
NAID 40020021459

Related Pictures

★リンクテーブル★

リンク元	「部位特異的突然変異誘発」「site-specific mutagenesis」
関連記事	「direct」「sit」「site」「mutagenesis」「directed mutagenesis」