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Site-directed mutagenesis
From Wikipedia, the free encyclopedia
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 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.
Contents
1 History
2 Basic mechanism
3 Approaches in site-directed mutagenesis
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 See also
6 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 N
4
-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 X174 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
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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. The 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 in site-directed mutagenesis
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 1987, 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
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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 separate 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.
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,
[14]
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.
[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 long
homologous 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 has
improved or special properties.
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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.
[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: 265
294. 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 base
substitutions 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 an
extracistronic 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): 181
189. PMC 356229 (//www.ncbi.nlm.nih.gov/pmc/articles/PMC356229). PMID 4940243
(//www.ncbi.nlm.nih.gov/pubmed/4940243).
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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
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9. ^ Marshall H. Edgell, Clyde A. Hutchison, III, and Morton Sclair (1972). "Specific Endonuclease R Fragments
of 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 a
specific 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 two
single-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 and
chromosome 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 be
resistant 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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