site-directed mutagenesis relies on mut proteins to cause base replacements
Site - directed mutagenesis - wikipedia Site - directed mutagenesis Jump to : navigation , search 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 . Contents ( hide ) 1 History 2 Basic mechanism 3 Approaches 3.1 Kunkel 's method 3.2 Cassette mutagenesis 3.3 PCR site - directed mutagenesis 3.4 Whole plasmid mutagenesis 3.5 In vivo site - directed mutagenesis methods 4 Applications 5 Gene synthesis 6 See also 7 References 8 External links History ( edit ) 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 N - 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 . Basic mechanism ( edit ) 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 ( edit ) 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 . Kunkel 's method ( edit ) 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 ( 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 ( edit ) 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 . PCR site - directed mutagenesis ( edit ) 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 can simplify the process . Whole plasmid mutagenesis ( edit ) 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 vivo site - directed mutagenesis methods ( edit ) 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 Applications ( edit ) 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 . 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 . Gene synthesis ( edit ) 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 . See also ( edit ) Directed mutagenesis References ( edit ) Jump up ^ Hsu PD , Lander ES , Zhang F ( June 2014 ) . `` Development and applications of CRISPR - Cas9 for genome engineering '' . Cell. 157 ( 6 ) : 1262 -- 78 . PMC 4343198 . PMID 24906146 . doi : 10.1016 / j. cell. 2014.05. 010 . Jump up ^ Kilbey , B.J. ( 1995 ) . `` Charlotte Auerbach ( 1899 - 1994 ) '' . Genetics . 141 ( 1 ) : 1 -- 5 . PMC 1206709 . PMID 8536959 . Jump up ^ Shortle , D. ; Dimaio , D. ; Nathans , D. ( 1981 ) . `` Directed Mutagenesis '' . Annual Review of Genetics . 15 : 265 -- 294 . PMID 6279018 . doi : 10.1146 / annurev. ge. 15.120181. 001405 . Jump up ^ 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 . Jump up ^ 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 . Jump up ^ 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 . PMC 392513 . PMID 209457 . doi : 10.1073 / pnas. 75.5. 2170 . Jump up ^ RA Flavell ; DL Sabo ; EF 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 USA . 72 ( 1 ) : 367 -- 371 . PMC 432306 . PMID 47176 . doi : 10.1073 / pnas. 72.1. 367 . Jump up ^ 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 . PMID 712841 . doi : 10.1016 / 0022 - 2836 ( 78 ) 90303 - 0 . Jump up ^ 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 . Jump up ^ 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 . CS1 maint : Multiple names : authors list ( link ) Jump up ^ 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 . Jump up ^ Braman , Jeff , ed. ( 2002 ) . In Vitro Mutagenesis Protocols . Methods in Molecular Biology . 182 ( 2nd ed . ) . Humana Press . ISBN 978 - 0896039100 . Jump up ^ Kunkel TA. ( 1985 ) . `` Rapid and efficient site - specific mutagenesis without phenotypic selection . '' ( PDF ) . Proceedings of the National Academy of Sciences . 82 ( 2 ) : 488 -- 92 . PMC 397064 . PMID 3881765 . doi : 10.1073 / pnas. 82.2. 488 . Jump up ^ Wells , J.A. ; Estell , D.A. ( 1988 ) . `` Subtilisin -- an enzyme designed to be engineered '' . Trends in Biochemical Sciences . 13 ( 8 ) : 291 -- 297 . PMID 3154281 . doi : 10.1016 / 0968 - 0004 ( 88 ) 90121 - 1 . Jump up ^ 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 . ISSN 1471 - 2105 . PMC 3644487 . PMID 23522286 . doi : 10.1186 / 1471 - 2105 - 14 - 105 . Jump up ^ 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 . CS1 maint : Multiple names : authors list ( link ) ^ Jump up to : 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 . PMC 2711942 . PMID 19566935 . doi : 10.1186 / 1472 - 6750 - 9 - 61 . Jump up ^ 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 . PMID 16793410 . doi : 10.1016 / S0076 - 6879 ( 05 ) 09019 - 1 . Jump up ^ Storici F. ; Resnick MA ( 2003 ) . `` Delitto perfetto targeted mutagenesis in yeast with oligonucleotides '' . Genetic Engineering . 25 : 189 -- 207 . PMID 15260239 . Jump up ^ 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 . Jump up ^ 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 . Jump up ^ Yury E. Khudyakov , Howard A. Fields , ed. ( 25 September 2002 ) . Artificial DNA : Methods and Applications . CRC Press . p. 13 . ISBN 9781420040166 . External links ( edit ) Library resources about Site - directed mutagenesis Resources in your library Resources in other libraries Nobel Lecture on Invention of Site - Directed Mutagenesis OpenWetWare Diagram summarizing site - directed mutagenesis Retrieved from `` https://en.wikipedia.org/w/index.php?title=Site-directed_mutagenesis&oldid=802188715 '' Categories : Molecular genetics Mutagenesis Protein engineering Hidden categories : CS1 maint : Multiple names : authors list Talk Contents About Wikipedia Deutsch Dolnoserbski Español Français Italiano עברית Português Русский Українська 中文 Edit links This page was last edited on 24 September 2017 , at 15 : 22 . Text is available under the Creative Commons Attribution - ShareAlike License ; additional terms may apply . By using this site , you agree to the Terms of Use and Privacy Policy . Wikipedia ® is a registered trademark of the Wikimedia Foundation , Inc. , a non-profit organization . About Wikipedia
1. Site-directed mutagenesis
2. DNA sequence modification
3. Protein engineering
4. Oligonucleotide-directed mutagenesis
5. CRISPR/Cas9 technology