Site-directed mutagenesis
Encyclopedia
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology
technique in which a mutation
is created at a defined site in a DNA
molecule. In general, this form of mutagenesis
requires that the wild type
gene sequence
be known. It is commonly used in protein engineering
.
using N4-hydroxycytidine which induces transition of GC to AT. These methods of mutagenesis however are limited by the kind of mutation they can achieve.
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 oligonucleotide
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
in October 1993 with Kary B. Mullis, who invented polymerase chain reaction
.
of a short DNA primer which is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation
), deletion or insertion
, containing the desired mutation such as a base change. This synthetic primer is complementary to the template DNA around the base change so it can hybridize with the DNA containing the gene of interest. The single-stranded 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.
The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favors the methylated template DNA. Many approaches have since been developed to improve the efficiency of mutagenesis.
and uracil deglycosidase
. The dUTPase deficiency prevents the breakdown of dUTP, a nucleotide
that normally replaces dTTP in RNA
, resulting in an abundance of dUTP; the uracil deglycosidase deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-mutant E. coli replicates the vector DNA, its enzymatic machinery may therefore misincorporates dUTP instead of dTTP, resulting in DNA which contains some uracils. This copy is extracted
, and then used as template for mutagenesis. An oligonucleotide
containing the desired mutation is use for primer extension. The heteroduplex DNA formed may be chimeric containing one strand unmutated and containing UTP, and the other strand mutated and containing dTTP. The DNA is then first treated with uracil deglycosidase which removes the uracil in the template, then with alkali which degrades the template DNA as it has its uracil removed making it sensitive to alkali. The resulting DNA therefore consists of only the mutated strand.
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 cuts 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.
with oligonucleotide
"primers
" that contain the desired mutation. As the primers are the ends of newly-synthesized strands, by engineering a mis-match during the first cycle in binding the template DNA strand, a mutation can be introduced. Because PCR employs exponential growth, after a sufficient number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques. There are many variations of the same technique.
reaction using a high-fidelity DNA polymerase such as pfu polymerase
. However, unlike PCR, this process does not amplify the DNA exponentially, but linearly (it is therefore inaccurate to describe it as a PCR reaction since there is no chain reaction). The reaction generates a nicked
, circular DNA which is relaxed. 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 Escherichia coli
would be methylated; the template plasmid which is biosynthesized in E. coli will therefore be digested, while the mutated plasmid is generated in vitro and is therefore unmethylated would be left undigested.
protein that has improved or special properties.
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 proteins 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, inactivating the protein in the process. This methionine may be replaced by alanine, thereby making the protein active in the presence of bleach.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
technique in which a mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
is created at a defined site in a DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
molecule. In general, this form of mutagenesis
Mutagenesis
Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...
requires that the wild type
Wild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
gene sequence
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
be known. It is commonly used in protein engineering
Protein engineering
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles....
.
History
Early attempts at mutagenesis were non-site specific using radiation or chemical mutagens. 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 1973 in the laboratory of Charles WeissmannCharles Weissmann
Charles Weissmann is a Hungarian-born Swiss molecular biologist.Weissmann went to Zurich University and obtained his MD in 1956 and Ph. D. in Organic Chemistry in 1961. Weissmann was director of the Institute for Molecular Biology in Zurich, President of the Roche Research Foundation and...
using N4-hydroxycytidine which induces transition of GC to AT. These methods of mutagenesis however are limited by the kind of mutation they can achieve.
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
Michael Smith (chemist)
Michael Smith, CC, OBC, FRS was a British-born Canadian biochemist who won the 1993 Nobel Prize for Chemistry.-Biography:...
in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
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
Nobel Prize in Chemistry
The Nobel Prize in Chemistry is awarded annually by the Royal Swedish Academy of Sciences to scientists in the various fields of chemistry. It is one of the five Nobel Prizes established by the will of Alfred Nobel in 1895, awarded for outstanding contributions in chemistry, physics, literature,...
in October 1993 with Kary B. Mullis, who invented polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
.
Basic mechanism
The basic procedure requires the synthesisOligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
of a short DNA primer which is complementary to the template DNA around the site where the mutation is to be introduced. The mutation may be a single base change (a point mutation
Point mutation
A point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
), deletion or insertion
Insertion (genetics)
In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping...
, containing the desired mutation such as a base change. This synthetic primer is complementary to the template DNA around the base change so it can hybridize with the DNA containing the gene of interest. The single-stranded primer is then extended using a DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
, 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
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
. Finally, mutants are selected.
The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture may contain both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favors the methylated template DNA. 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, although most of them are now rarely used in laboratories since the early 2000s as newer techniques allow for simpler and easier way of introducing site-specific mutation into genes.Kunkel's method
In 1987 Kunkel et al. introduced a technique which reduces the need to select for the mutants. The vector DNA to be mutated is inserted into an E. coli strain deficient in two enzymes, dUTPaseDUT (gene)
DUTP pyrophosphatase, also known as DUT, is an enzyme which in humans is encoded by the DUT gene.- Function :This gene encodes an essential enzyme of nucleotide metabolism. The encoded protein forms a ubiquitous, homotrimeric enzyme that hydrolyzes dUTP to dUMP and pyrophosphate...
and uracil deglycosidase
Uracil-DNA glycosylase
Uracil-DNA glycosylase, also known as UNG or UDG, is a human gene though orthologs exist ubiquitously among prokaryotes and eukaryotes and even in some DNA viruses. The first uracil DNA-glycosylase was isolated from Escherichia coli....
. The dUTPase deficiency prevents the breakdown of dUTP, a nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
that normally replaces dTTP in RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, resulting in an abundance of dUTP; the uracil deglycosidase deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-mutant E. coli replicates the vector DNA, its enzymatic machinery may therefore misincorporates dUTP instead of dTTP, resulting in DNA which contains some uracils. This copy is extracted
DNA extraction
DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction:...
, and then used as template for mutagenesis. An oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
containing the desired mutation is use for primer extension. The heteroduplex DNA formed may be chimeric containing one strand unmutated and containing UTP, and the other strand mutated and containing dTTP. The DNA is then first treated with uracil deglycosidase which removes the uracil in the template, then with alkali which degrades the template DNA as it has its uracil removed making it sensitive to alkali. The resulting DNA therefore consists of only 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 enzymeRestriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
at a site in the plasmid and subsequent ligation
Ligation
Ligation may refer to:* In molecular biology, the covalent linking of two ends of DNA molecules using DNA ligase* In medicine, the making of a ligature * Chemical ligation, the production of peptides from amino acids...
of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid. Usually the restriction enzymes that cuts 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 same result can be accomplished using polymerase chain reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
with oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
"primers
Primer (molecular biology)
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...
" that contain the desired mutation. As the primers are the ends of newly-synthesized strands, by engineering a mis-match during the first cycle in binding the template DNA strand, a mutation can be introduced. Because PCR employs exponential growth, after a sufficient number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques. There are many variations of the same technique.
Plasmid mutagenesis
For plasmid manipulations, other site-directed mutagenesis techniques have been largely supplanted by a PCR-like technique which is highly efficient but relatively simple, easy to use, and commercially available as a kit in products such as QuikChange. In this technique, a pair of complementary mutagenic primers are used to amplify the entire plasmid in a thermocyclingThermal cycler
The thermal cycler is a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction process. The device has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted...
reaction using a high-fidelity DNA polymerase such as pfu polymerase
Pfu DNA polymerase
Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo to replicate the organism's DNA...
. However, unlike PCR, this process does not amplify the DNA exponentially, but linearly (it is therefore inaccurate to describe it as a PCR reaction since there is no chain reaction). The reaction generates a nicked
Nick (DNA)
A nick is a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand typically through damage or enzyme action. Nick allows for much needed release of torsion in the strand....
, circular DNA which is relaxed. The template DNA must be eliminated by enzymatic digestion with a restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
such as DpnI which is specific for methylated DNA. All DNA produced from Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
would be methylated; the template plasmid which is biosynthesized in E. coli will therefore be digested, while the mutated plasmid is generated in vitro and is therefore unmethylated would be left undigested.
In vivo site-directed mutagenesis methods
- Delitto perfettoDelitto perfettoDelitto perfetto is a genetic technique for in vivo site-directed mutagenesis in yeast. This name is an Italian term for perfect deletion and is also an idiom for perfect murder...
- 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
Site-directed mutagenesis is used to generate mutations that may produce rationally designedRational design
In chemical biology and biomolecular engineering, rational design is the strategy of creating new molecules with a certain functionality, based upon the ability to predict how the molecule's structure will affect its behavior through physical models...
protein that has improved or special properties.
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 proteins that are tailored for a specific application. For example, commonly-used laundry detergents may contain subtilisin
Subtilisin
Subtilisin is a non-specific protease initially obtained from Bacillus subtilis.Subtilisins belong to subtilases, a group of serine proteases that initiate the nucleophilic attack on the peptide bond through a serine residue at the active site. They are physically and chemically...
whose wild-type form has a methionine that can be oxidized by bleach, inactivating the protein in the process. This methionine may be replaced by alanine, thereby making the protein active in the presence of bleach.
