site-directed mutagenesis - wikipedia, the free encyclopedia

4/27/2014 Site-directed mutagenesis - Wikipedia, the free encyclopedia

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Site-directed mutagenesisFrom Wikipedia, the free encyclopedia

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changesto the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis oroligonucleotide-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 mutation into aDNA sequence. However, with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is nowoccasionally used as an alternative to site-directed mutagenesis.

Contents

1 History

2 Basic mechanism

3 Approaches in site-directed mutagenesis

3.1 Kunkel's method

3.2 Cassette mutagenesis3.3 PCR site-directed mutagenesis

3.4 Whole plasmid mutagenesis

3.5 In vivo site-directed mutagenesis methods

4 Applications

5 See also6 References

7 External links

History

Early attempts at mutagenesis using radiation or chemical mutagens were non-site-specific.[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

1973 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 theycan 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 X174 and restriction nucleases.[8][9] Hutchison later produced with his collaboratorMichael 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, MichaelSmith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerasechain reaction.

Basic mechanism



The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desiredmutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNAin 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 restof the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vectorand 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. The resultingmixture contains both the original unmutated template as well as the mutant strand, producing a mixed populationof mutant and non-mutant progenies. Furthermore the template used is methylated while the mutant strand isunmethylated, and the mutants may be counter-selected due to presence of mismatch repair system that favorsthe methylated template DNA, resulting in fewer mutants. Many approaches have since been developed toimprove the efficiency of mutagenesis.

Approaches in site-directed mutagenesis

A large number of methods are available to effect site-directed mutagenesis,[11] although most of them are nowrarely used in laboratories since the early 2000s, as newer techniques allow for simpler and easier ways ofintroducing site-specific mutation into genes.

Kunkel's method

In 1987, Thomas Kunkel introduced a technique that reduces the need to select for the mutants.[12] The DNAfragment to be mutated is inserted into a phagemid such as M13mp18/19 and is then transformed into an E. colistrain deficient in two enzymes, dUTPase (dut) and uracil deglycosidase (ung). Both enzymes are part of aDNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination ofdCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP inthe cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly synthesized DNA. As thedouble-mutant E. coli replicates the phage DNA, its enzymatic machinery may, therefore, misincorporate dUTPinstead of dTTP, resulting in single-strand DNA that contains some uracils (ssUDNA). The ssUDNA isextracted 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 thatforms consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP. TheDNA 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 mutatedstrand.

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 arestriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotidescontaining the mutation in the gene of interest to the plasmid. Usually, the restriction enzymes that cut at theplasmid and the oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to oneanother. This method can generate mutants at close to 100% efficiency, but is limited by the availability ofsuitable 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 witholigonucleotide "primers", such that a larger fragment may be generated, covering two convenient restrictionsites. The exponential amplification in PCR produces a fragment containing the desired mutation in sufficientquantity to be separate from the original, unmutated plasmid by gel electrophoresis, which may then be insertedin the original context using standard recombinant molecular biology techniques. There are many variations of thesame technique. The simplest method places the mutation site toward one of the ends of the fragment wherebyone of two oligonucleotides used for generating the fragment contains the mutation. This involves a single step ofPCR, but still has the inherent problem of requiring a suitable restriction site near the mutation site unless a verylong primer is used. Other variations, therefore, employ three or four oligonucleotides, two of which may benon-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can bedigested and ligated into a plasmid, whereas the mutagenic oligonucleotide may be complementary to a locationwithin that fragment well away from any convenient restriction site. These methods require multiple steps of PCRso 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 supplanted largely bytechniques 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,[14] wherein a pair of complementary mutagenic primersare used to amplify the entire plasmid in a thermocycling reaction using a high-fidelity non-strand-displacingDNA polymerase such as pfu polymerase. The reaction generates a nicked, circular DNA. The template DNAmust be eliminated by enzymatic digestion with a restriction enzyme such as DpnI, which is specific formethylated DNA. All DNA produced from most Escherichia coli strains would be methylated; the templateplasmid that is biosynthesized in E. coli will, therefore, be digested, while the mutated plasmid, which isgenerated in vitro and is therefore unmethylated, would be left undigested. Note that, in these double-strandplasmid mutagenesis methods, while the thermocycling reaction may be used, the DNA need not beexponentially amplified as in a PCR. Instead, the amplification is linear, and it is therefore inaccurate to describethem as a PCR, since there is no chain reaction.

Note that pfu polymerase can become strand-displacing at higher extension temperature (70 C) which canresult in the failure of the experiment, therefore the extension reaction should be performed at the recommendedtemperature of 68 C. In some applications, this method has been observed to lead to insertion of multiple

copies of primers.[15] A variation of this method, called SPRINP, prevents this artifact and has been used in

different types of site directed mutagenesis.[15]

In vivo site-directed mutagenesis methods

Delitto perfetto[16]

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 longhomologous regions

In vivo site-directed mutagenesis with synthetic oligonucleotides[17]

Applications

Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that hasimproved or special properties.



Investigative tools specific mutations in DNA allow the function and properties of a DNA sequence or aprotein to be investigated in a rational approach.

Commercial applications Proteins may be engineered to produce mutant forms that are tailored for aspecific application. For example, commonly used laundry detergents may contain subtilisin, whose wild-typeform has a methionine that can be oxidized by bleach, significantly reducing the activity the protein in the

process.[18] 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.[19]

See also

Directed mutagenesis

References

1. ^ Kilbey, B. J. (1995). "Charlotte Auerbach (1899-1994)"

(//www.ncbi.nlm.nih.gov/pmc/articles/PMC1206709). Genetics 141 (1): 15. PMC 1206709(//www.ncbi.nlm.nih.gov/pmc/articles/PMC1206709). PMID 8536959(//www.ncbi.nlm.nih.gov/pubmed/8536959).

2. ^ Shortle, D.; Dimaio, D.; Nathans, D. (1981). "Directed Mutagenesis". Annual Review of Genetics 15: 265294. doi:10.1146/annurev.ge.15.120181.001405 (http://dx.doi.org/10.1146%2Fannurev.ge.15.120181.001405).PMID 6279018 (//www.ncbi.nlm.nih.gov/pubmed/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"

(//www.ncbi.nlm.nih.gov/pmc/articles/PMC369902). Molecular and Cellular Biology 2 (9): 10961103.PMC 369902 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC369902). PMID 6983647(//www.ncbi.nlm.nih.gov/pubmed/6983647).

4. ^ McHugh, G. L.; Miller, C. G. (1974). "Isolation and Characterization of Proline Peptidase Mutants of

Salmonella typhimurium" (//www.ncbi.nlm.nih.gov/pmc/articles/PMC245771). Journal of bacteriology 120 (1):364371. PMC 245771 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC245771). PMID 4607625(//www.ncbi.nlm.nih.gov/pubmed/4607625).

5. ^ D Shortle and D Nathans (1978). "Local mutagenesis: a method for generating viral mutants with basesubstitutions in preselected regions of the viral genome." (//www.ncbi.nlm.nih.gov/pmc/articles/PMC392513).

Proceedings of the National Academy of Sciences U S A. 75 (5): 21702174. PMC 392513(//www.ncbi.nlm.nih.gov/pmc/articles/PMC392513). PMID 209457(//www.ncbi.nlm.nih.gov/pubmed/209457).

6. ^ R A Flavell, D L Sabo, E F Bandle, and C Weissmann (1975). "Site-directed mutagenesis: effect of anextracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA"

(//www.ncbi.nlm.nih.gov/pmc/articles/PMC432306). Proc Natl Acad Sci U S A. 72 (1): 367371.doi:10.1073/pnas.72.1.367 (http://dx.doi.org/10.1073%2Fpnas.72.1.367). PMC 432306(//www.ncbi.nlm.nih.gov/pmc/articles/PMC432306). PMID 47176 (//www.ncbi.nlm.nih.gov/pubmed/47176).

7. ^ Willi Mller, Hans Weber, Franois Meyer, Charles Weissmann (1978). "Site-directed mutagenesis in DNA:Generation of point mutations in cloned globin complementary DNA at the positions corresponding to amino

acids 121 to 123". Journal of Molecular Biology 124 (2): 343358. doi:10.1016/0022-2836(78)90303-0(http://dx.doi.org/10.1016%2F0022-2836%2878%2990303-0). PMID 712841(//www.ncbi.nlm.nih.gov/pubmed/712841).

8. ^ Hutchison Ca, 3.; Edgell, M. H. (1971). "Genetic Assay for Small Fragments of Bacteriophage X174

Deoxyribonucleic Acid" (//www.ncbi.nlm.nih.gov/pmc/articles/PMC356229). Journal of Virology 8 (2): 181189. PMC 356229 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC356229). PMID 4940243(//www.ncbi.nlm.nih.gov/pubmed/4940243).



External links

Nobel Lecture on Invention of Site-Directed Mutagenesis

(http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/smith-lecture.html)

OpenWetWare (http://openwetware.org/wiki/Site-directed_mutagenesis)Diagram summarizing site-directed mutagenesis

(http://bioweb.wku.edu/courses/biol350/Mutagenesis18/Images/Ch9E2.gif)

Retrieved from "http://en.wikipedia.org/w/index.php?title=Site-directed_mutagenesis&oldid=605264036"Categories: Molecular genetics Mutagenesis Protein engineering

This page was last modified on 22 April 2014 at 07:45.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

9. ^ Marshall H. Edgell, Clyde A. Hutchison, III, and Morton Sclair (1972). "Specific Endonuclease R Fragmentsof Bacteriophage X174 Deoxyribonucleic Acid" (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC356341/).

Journal of Virology 9 (4): 574582. PMC 356341 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC356341).PMID 4553678 (//www.ncbi.nlm.nih.gov/pubmed/4553678).

10. ^ Hutchison CA, Phillips S, Edgell MH, Gillam S, Jahnke P, Smith M (September 1978). "Mutagenesis at aspecific position in a DNA sequence" (http://www.jbc.org/content/253/18/6551.full.pdf) (PDF). J. Biol. Chem.

253 (18): 655160. PMID 681366 (//www.ncbi.nlm.nih.gov/pubmed/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."(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC397064/pdf/pnas00342-0237.pdf). Proceedings of the

National Academy of Sciences U S A. 82 (2): 48892. PMC 397064(//www.ncbi.nlm.nih.gov/pmc/articles/PMC397064). PMID 3881765(//www.ncbi.nlm.nih.gov/pubmed/3881765).

13. ^ Wells, J. A.; Estell, D. A. (1988). "Subtilisin--an enzyme designed to be engineered". Trends in Biochemical

Sciences 13 (8): 291297. doi:10.1016/0968-0004(88)90121-1 (http://dx.doi.org/10.1016%2F0968-0004%2888%2990121-1). PMID 3154281 (//www.ncbi.nlm.nih.gov/pubmed/3154281).

14. ^ Papworth, C., Bauer, J. C., Braman, J. and Wright, D. A. (1996). "Site-directed mutagenesis in one day with

>80% efficiency.". Strategies 9 (3): 34.

15. ^a b Edelheit, O; Hanukoglu, A; Hanukoglu, I (2009). "Simple and efficient site-directed mutagenesis using twosingle-primer reactions in parallel to generate mutants for protein structure-function studies"

(http://www.biomedcentral.com/1472-6750/9/61). BMC Biotechnol 9: 61. doi:10.1186/1472-6750-9-61(http://dx.doi.org/10.1186%2F1472-6750-9-61). PMID 19566935(//www.ncbi.nlm.nih.gov/pubmed/19566935).

16. ^ Storici F., Resnick MA. (2006). "The delitto perfetto approach to in vivo site-directed mutagenesis andchromosome rearrangements with synthetic oligonucleotides in yeast."

(http://www.ncbi.nlm.nih.gov/pubmed/16793410). Methods in Enzymology 409: 32945. doi:10.1016/S0076-6879(05)09019-1 (http://dx.doi.org/10.1016%2FS0076-6879%2805%2909019-1). PMID 16793410(//www.ncbi.nlm.nih.gov/pubmed/16793410).

17. ^ Storici F., Resnick MA (2003). "Delitto perfetto targeted mutagenesis in yeast with oligonucleotides". Genetic

Engineering 25: 189207. PMID 15260239 (//www.ncbi.nlm.nih.gov/pubmed/15260239).

18. ^ Stauffer CE, Etson D (October 10, 1969). "The effect on subtilisin activity of oxidizing a methionine residue"

(http://www.jbc.org/content/244/19/5333). Journal of Biological Chemistry 244 (19): 53338. PMID 5344139(//www.ncbi.nlm.nih.gov/pubmed/5344139).

19. ^ Estell DA, Graycar TP, Wells JA (10 June 1985). "Engineering an enzyme by site-directed mutagenesis to beresistant to chemical oxidation" (http://www.jbc.org/content/260/11/6518.long). Journal of Biological

Chemistry 260 (11): 651821. PMID 3922976 (//www.ncbi.nlm.nih.gov/pubmed/3922976).



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