Site-directed mutagenesis is a
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
method that is used to make specific and intentional mutating changes to the
DNA sequence of a
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
and any
gene product
A gene product is the biochemical material, either RNA or protein, resulting from expression of a gene. A measurement of the amount of gene product is sometimes used to infer how active a gene is. Abnormal amounts of gene product can be correlate ...
s. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of
DNA,
RNA, and
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
molecules, and for
protein engineering.
Site-directed mutagenesis is one of the most important laboratory techniques for creating
DNA libraries by introducing mutations into DNA sequences. There are numerous methods for achieving site-directed mutagenesis, but with decreasing costs of
oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpen ...
,
artificial gene synthesis
Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides '' de novo''. Unlike DNA synthesis in living cells, artificial gene synthesis d ...
is now occasionally used as an alternative to site-directed mutagenesis. Since 2013, the 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'' with relative ease.
History
Early attempts at
mutagenesis
Mutagenesis () is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using lab ...
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
A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...
, examples of such chemicals are
aminopurine
2-Aminopurine, a purine analog of guanine and adenine, is a fluorescent molecular marker used in nucleic acid research. It most commonly pairs with thymine as an adenine-analogue, but can also pair with cytosine
Cytosine () ( symbol C or Cyt) ...
,
nitrosoguanidine, and
bisulfite
The bisulfite ion (IUPAC-recommended nomenclature: hydrogensulfite) is the ion . Salts containing the ion are also known as "sulfite lyes". Sodium bisulfite is used interchangeably with sodium metabisulfite (Na2S2O5). Sodium metabisulfite disso ...
. Site-directed mutagenesis was achieved in 1974 in the laboratory of
Charles Weissmann
Charles Weissmann (born 14 October 1931) is a Hungarian-Swiss molecular biologist. Weissmann is particularly known for the first cloning and expression of interferon and his contributions to the unraveling of the molecular genetics of neurogener ...
using a nucleotide analogue N
4-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 nuclease
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...
s. Hutchison later produced with his collaborator
Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
s 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
)
, image = Nobel Prize.png
, alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
in October 1993 with
Kary B. Mullis, who invented
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
.
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
Hybridization (or hybridisation) may refer to:
*Hybridization (biology), the process of combining different varieties of organisms to create a hybrid
*Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals
*Nu ...
with the DNA in the gene of interest. The mutation may be a single base change (a
point mutation
A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...
), multiple base changes,
deletion, or
insertion. The single-strand primer is then extended using a
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell in a vector and
cloned
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, ...
. Finally, mutants are selected by
DNA sequencing to check that they contain the desired mutation.
Approaches
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
In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These ...
while the mutant strand is unmethylated, and the mutants may be counter-selected due to presence of
mismatch repair
DNA mismatch repair (MMR) is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.
Mismatch ...
system that favors the methylated template DNA, resulting in fewer mutants. Many approaches have since been developed to improve the efficiency of mutagenesis.
A large number of methods are available to effect site-directed mutagenesis, although most of them have rarely been 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. The DNA fragment to be mutated is inserted into a
phagemid
A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin of replication derived from bacteriophage. Unlike commonly u ...
such as
M13mp18/19 and is then transformed into an ''
E. coli'' strain deficient in two enzymes,
dUTPase (''
dut'') and
uracil deglycosidase (''udg''). Both enzymes are part of a
DNA repair
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...
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
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
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 ''udg'' 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. It involves the cleavage by a
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
at a site in the plasmid and subsequent
ligation
Ligation may refer to:
* Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules
* In medicine, the making of a ligature (tie)
* Chemical ligation, the production of peptides from amino acids
* Tubal ligation, a meth ...
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
![Site-directed mutagenesis library cloning steps](https://upload.wikimedia.org/wikipedia/commons/7/77/Site-directed_mutagenesis_library_cloning_steps.svg)
The limitation of restriction sites in cassette mutagenesis may be overcome using
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
with
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
"
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
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
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
such as ''Dpn''I, which is specific for methylated DNA. All DNA produced from most ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
'' 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.
Other techniques such as scanning mutagenesis of oligo-directed targets (SMOOT) can semi-randomly combine mutagenic oligonucleotides in plasmid mutagenesis. This technique can create plasmid mutagenesis libraries ranging from single mutations to comprehensive codon mutagenesis across an entire gene.
''In vivo'' site-directed mutagenesis methods
*''
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
CRISPR
Since 2013, the development of
CRISPR-Cas9 technology has allowed for the efficient introduction of various mutations into the genome of a wide variety of organisms. The method does not require a transposon insertion site, leaves no marker, and its efficiency and simplicity has made it the preferred method for
genome editing
Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts ...
.
Applications
![Site saturation mutagenesis](https://upload.wikimedia.org/wikipedia/commons/f/f8/Site_saturation_mutagenesis.svg)
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. Furthermore, single amino-acid changes by site-directed mutagenesis in proteins can help understand the importance of post-translational modifications. For instance changing a particular serine (phosphoacceptor) to an alanine (phospho-non-acceptor) in a substrate protein blocks the attachment of a phosphate group, thereby allows the phosphorylation to be investigated. This approach has been used to uncover the phosphorylation of the protein
CBP CBP may refer to:
Business parks
* Cebu Business Park, a central business district in Cebu City, Philippines
* Changi Business Park, an eco-friendly industrial park in Singapore
* Chiswick Business Park, a business park in Gunnersbury, West London
...
by the kinase
HIPK2
Homeodomain-interacting protein kinase 2 is an enzyme that in humans is encoded by the ''HIPK2'' gene. HIPK2 can be categorized as a Serine/Threonine Protein kinase, specifically one that interacts with homeodomain transcription factors. It belong ...
Another comprehensive approach is site
saturation mutagenesis
Site saturation mutagenesis (SSM), or simply site saturation, is a random mutagenesis technique used in protein engineering, in which a single codon or set of codons is substituted with all possible amino acids at the position. There are many v ...
where one
codon or a set of codons may be substituted with all possible
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha a ...
s at the specific positions.
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
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
*
Directed mutagenesis
*
Phi value analysis Phi value analysis,
\phi
analysis, or \phi-value analysis is an experimental protein engineering technique for studying the structure of the folding transition state of small protein domains that fold in a two-state manner. The structure of the f ...
References
External links
{{Library resources box
, onlinebooks=no
, by=no
, lcheading=Site-specific mutagenesis
Nobel Lecture on Invention of Site-Directed MutagenesisOpenWetWareDiagram summarizing site-directed mutagenesis
Genetics techniques
Molecular genetics
Mutagenesis
Protein engineering