Mutagenesis (molecular biology technique)

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Types of mutations that can be introduced by random, site-directed, combinatorial, or insertional mutagenesis. DE Mutations.png
Types of mutations that can be introduced by random, site-directed, combinatorial, or insertional mutagenesis.

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

Contents

Many methods of mutagenesis exist today. Initially, the kind of mutations artificially induced in the laboratory were entirely random using mechanisms such as UV irradiation. Random mutagenesis cannot target specific regions or sequences of the genome; however, with the development of site-directed mutagenesis, more specific changes can be made. Since 2013, development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has allowed for the editing or mutagenesis of a genome in vivo. [1] Site-directed mutagenesis has proved useful in situations that random mutagenesis is not. Other techniques of mutagenesis include combinatorial and insertional mutagenesis. Mutagenesis that is not random can be used to clone DNA, [2] investigate the effects of mutagens, [3] and engineer proteins. [4] It also has medical applications such as helping immunocompromised patients, research and treatment of diseases including HIV and cancers, and curing of diseases such as beta thalassemia. [5]

Random mutagenesis

How DNA libraries generated by random mutagenesis sample sequence space. The amino acid substituted into a given position is shown. Each dot or set of connected dots is one member of the library. Error-prone PCR randomly mutates some residues to other amino acids. Alanine scanning replaces each residue of the protein with alanine, one-by-one. Site saturation substitutes each of the 20 possible amino acids (or some subset of them) at a single position, one-by-one. How random DNA libraries sample sequence space.pdf
How DNA libraries generated by random mutagenesis sample sequence space. The amino acid substituted into a given position is shown. Each dot or set of connected dots is one member of the library. Error-prone PCR randomly mutates some residues to other amino acids. Alanine scanning replaces each residue of the protein with alanine, one-by-one. Site saturation substitutes each of the 20 possible amino acids (or some subset of them) at a single position, one-by-one.

Early approaches to mutagenesis relied on methods which produced entirely random mutations. In such methods, cells or organisms are exposed to mutagens such as UV radiation or mutagenic chemicals, and mutants with desired characteristics are then selected. Hermann Muller discovered in 1927 that X-rays can cause genetic mutations in fruit flies, [6] and went on to use the mutants he created for his studies in genetics. [7] For Escherichia coli , mutants may be selected first by exposure to UV radiation, then plated onto an agar medium. The colonies formed are then replica-plated, one in a rich medium, another in a minimal medium, and mutants that have specific nutritional requirements can then be identified by their inability to grow in the minimal medium. Similar procedures may be repeated with other types of cells and with different media for selection.

A number of methods for generating random mutations in specific proteins were later developed to screen for mutants with interesting or improved properties. These methods may involve the use of doped nucleotides in oligonucleotide synthesis, or conducting a PCR reaction in conditions that enhance misincorporation of nucleotides (error-prone PCR), for example by reducing the fidelity of replication or using nucleotide analogues. [8] A variation of this method for integrating non-biased mutations in a gene is sequence saturation mutagenesis. [9] PCR products which contain mutation(s) are then cloned into an expression vector and the mutant proteins produced can then be characterised.

In animal studies, alkylating agents such as N-ethyl-N-nitrosourea (ENU) have been used to generate mutant mice. [10] [11] Ethyl methanesulfonate (EMS) is also often used to generate animal, plant, and virus mutants. [12] [13] [14]

In a European Union law (as 2001/18 directive), this kind of mutagenesis may be used to produce GMOs but the products are exempted from regulation: no labeling, no evaluation. [15]

Site-directed mutagenesis

Prior to the development site-directed mutagenesis techniques, all mutations made were random, and scientists had to use selection for the desired phenotype to find the desired mutation. Random mutagenesis techniques has an advantage in terms of how many mutations can be produced; however, while random mutagenesis can produce a change in single nucleotides, it does not offer much control as to which nucleotide is being changed. [5] Many researchers therefore seek to introduce selected changes to DNA in a precise, site-specific manner. Early attempts uses analogs of nucleotides and other chemicals were first used to generate localized point mutations. [16] Such chemicals include aminopurine, which induces an AT to GC transition, [17] while nitrosoguanidine, [18] bisulfite, [19] and N4-hydroxycytidine may induce a GC to AT transition. [20] [21] These techniques allow specific mutations to be engineered into a protein; however, they are not flexible with respect to the kinds of mutants generated, nor are they as specific as later methods of site-directed mutagenesis and therefore have some degree of randomness. Other technologies such as cleavage of DNA at specific sites on the chromosome, addition of new nucleotides, and exchanging of base pairs it is now possible to decide where mutations can go. [11] [8]

Simplified diagram of the site directed mutagenic technique using pre-fabricated oligonucleotides in a primer extension reaction with DNA polymerase Site Directed Mutagenesis.png
Simplified diagram of the site directed mutagenic technique using pre-fabricated oligonucleotides in a primer extension reaction with DNA polymerase

Current techniques for site-specific mutation originates from the primer extension technique developed in 1978. Such techniques commonly involve using pre-fabricated mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation or deletion or insertion of small stretches of DNA at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process. [3]

Newer and more efficient methods of site directed mutagenesis are being constantly developed. For example, a technique called "Seamless ligation cloning extract" (or SLiCE for short) allows for the cloning of certain sequences of DNA within the genome, and more than one DNA fragment can be inserted into the genome at once. [2]

Site directed mutagenesis allows the effect of specific mutation to be investigated. There are numerous uses; for example, it has been used to determine how susceptible certain species were to chemicals that are often used In labs. The experiment used site directed mutagenesis to mimic the expected mutations of the specific chemical. The mutation resulted in a change in specific amino acids and the effects of this mutation were analyzed. [3]

Site saturation mutagenesis is a type of site-directed mutagenesis. This image shows the saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants of the isoleucine at position 5 are shown below. Site saturation mutagenesis.svg
Site saturation mutagenesis is a type of site-directed mutagenesis. This image shows the saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants of the isoleucine at position 5 are shown below.

The site-directed approach may be done systematically in such techniques as alanine scanning mutagenesis, whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein. [22] Another comprehensive approach is site saturation mutagenesis where one codon or a set of codons may be substituted with all possible amino acids at the specific positions. [23] [24]

Combinatorial mutagenesis

Combinatorial mutagenesis is a site-directed protein engineering technique whereby multiple mutants of a protein can be simultaneously engineered based on analysis of the effects of additive individual mutations. [25] It provides a useful method to assess the combinatorial effect of a large number of mutations on protein function. [26] Large numbers of mutants may be screened for a particular characteristic by combinatorial analysis. [25] In this technique, multiple positions or short sequences along a DNA strand may be exhaustively modified to obtain a comprehensive library of mutant proteins. [25] The rate of incidence of beneficial variants can be improved by different methods for constructing mutagenesis libraries. One approach to this technique is to extract and replace a portion of the DNA sequence with a library of sequences containing all possible combinations at the desired mutation site. The content of the inserted segment can include sequences of structural significance, immunogenic property, or enzymatic function. A segment may also be inserted randomly into the gene in order to assess structural or functional significance of a particular part of a protein. [25]

Insertional mutagenesis

The insertion of one or more base pairs, resulting in DNA mutations, is also known as insertional mutagenesis. [27] Engineered mutations such as these can provide important information in cancer research, such as mechanistic insights into the development of the disease. Retroviruses and transposons are the chief instrumental tools in insertional mutagenesis. Retroviruses, such as the mouse mammory tumor virus and murine leukemia virus, can be used to identify genes involved in carcinogenesis and understand the biological pathways of specific cancers. [28] Transposons, chromosomal segments that can undergo transposition, can be designed and applied to insertional mutagenesis as an instrument for cancer gene discovery. [28] These chromosomal segments allow insertional mutagenesis to be applied to virtually any tissue of choice while also allowing for more comprehensive, unbiased depth in DNA sequencing. [28]

Researchers have found four mechanisms of insertional mutagenesis that can be used on humans. the first mechanism is called enhancer insertion. Enhancers boost transcription of a particular gene by interacting with a promoter of that gene. This particular mechanism was first used to help severely immunocompromised patients I need of bone marrow. Gammaretroviruses carrying enhancers were then inserted into patients. The second mechanism is referred to as promoter insertion. Promoters provide our cells with the specific sequences needed to begin translation. Promoter insertion has helped researchers learn more about the HIV virus. The third mechanism is gene inactivation. An example of gene inactivation is using insertional mutagenesis to insert a retrovirus that disrupts the genome of the T cell in leukemia patients and giving them a specific antigen called CAR allowing the T cells to target cancer cells. The final mechanisms is referred to as mRNA 3' end substitution. Our genes occasionally undergo point mutations causing beta-thalassemia that interrupts red blood cell function. To fix this problem the correct gene sequence for the red blood cells are introduced and a substitution is made. [5]

Homologous recombination

Homologous recombination can be used to produce specific mutation in an organism. Vector containing DNA sequence similar to the gene to be modified is introduced to the cell, and by a process of recombination replaces the target gene in the chromosome. This method can be used to introduce a mutation or knock out a gene, for example as used in the production of knockout mice. [29]

CRISPR

Since 2013, the development of CRISPR-Cas9 technology has allowed for the efficient introduction of different types of 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. [30] [31]

Gene synthesis

As the cost of DNA oligonucleotide synthesis falls, artificial synthesis of a complete gene is now a viable method for introducing mutations into a gene. This method allows for extensive mutation at multiple sites, including the complete redesign of the codon usage of a gene to optimise it for a particular organism. [32]

See also

Related Research Articles

<span class="mw-page-title-main">Mutation</span> Alteration in the nucleotide sequence of a genome

In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis or other types of damage to DNA, which then may undergo error-prone repair, cause an error during other forms of repair, or cause an error during replication. Mutations may also result from insertion or deletion of segments of DNA due to mobile genetic elements.

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.

A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens can provide important information on gene function as well as the molecular events that underlie a biological process or pathway. While genome projects have identified an extensive inventory of genes in many different organisms, genetic screens can provide valuable insight as to how those genes function.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating 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.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

<span class="mw-page-title-main">Functional genomics</span> Field of molecular biology

Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional "candidate-gene" approach.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

Forward genetics is a molecular genetics approach of determining the genetic basis responsible for a phenotype. Forward genetics provides an unbiased approach because it relies heavily on identifying the genes or genetic factors that cause a particular phenotype or trait of interest.

<span class="mw-page-title-main">Insertion (genetics)</span> Type of mutation

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. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.

In molecular biology, insertional mutagenesis is the creation of mutations in DNA by the addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for research purposes in the lab.

<span class="mw-page-title-main">Gene targeting</span> Genetic technique that uses homologous recombination to change an endogenous gene

Gene targeting is a biotechnological tool used to change the DNA sequence of an organism. It is based on the natural DNA-repair mechanism of Homology Directed Repair (HDR), including Homologous Recombination. Gene targeting can be used to make a range of sizes of DNA edits, from larger DNA edits such as inserting entire new genes into an organism, through to much smaller changes to the existing DNA such as a single base-pair change. Gene targeting relies on the presence of a repair template to introduce the user-defined edits to the DNA. The user will design the repair template to contain the desired edit, flanked by DNA sequence corresponding (homologous) to the region of DNA that the user wants to edit; hence the edit is targeted to a particular genomic region. In this way Gene Targeting is distinct from natural homology-directed repair, during which the ‘natural’ DNA repair template of the sister chromatid is used to repair broken DNA. The alteration of DNA sequence in an organism can be useful in both a research context – for example to understand the biological role of a gene – and in biotechnology, for example to alter the traits of an organism.

Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

<span class="mw-page-title-main">Knockout rat</span> Type of genetically engineered rat

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

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 genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

References

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