Mutagenesis (molecular biology technique)#random mutagenesis

File:How random DNA libraries sample sequence space.pdf 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,{{cite journal | vauthors = Muller HJ | title = Artificial Transmutation of the Gene | journal = Science | volume = 66 | issue = 1699 | pages = 84–7 | date = July 1927 | pmid = 17802387 | doi = 10.1126/science.66.1699.84 | url = http://www.esp.org/foundations/genetics/classical/holdings/m/hjm-1927a.pdf | bibcode = 1927Sci....66...84M }} and went on to use the mutants he created for his studies in genetics.{{cite journal | vauthors = Crow JF, Abrahamson S | title = Seventy years ago: mutation becomes experimental | journal = Genetics | volume = 147 | issue = 4 | pages = 1491–6 | date = December 1997 | doi = 10.1093/genetics/147.4.1491 | pmid = 9409815 | pmc = 1208325 }} 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.{{cite book |url=https://books.google.com/books?id=5hoJ4d0-lvUC&pg=PA191 |title=Nucleic Acids in Chemistry and Biology| veditors = Blackburn GM |pages=191–192 |publisher=Royal Society of Chemistry |edition= 3rd |year= 2006 |isbn=978-0854046546 }} A variation of this method for integrating non-biased mutations in a gene is sequence saturation mutagenesis.{{cite journal | vauthors = Wong TS, Tee KL, Hauer B, Schwaneberg U | title = Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution | journal = Nucleic Acids Research | volume = 32 | issue = 3 | pages = 26e–26 | date = February 2004 | pmid = 14872057 | pmc = 373423 | doi = 10.1093/nar/gnh028 }} 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.{{cite journal | vauthors = Justice MJ, Noveroske JK, Weber JS, Zheng B, Bradley A | title = Mouse ENU mutagenesis | journal = Human Molecular Genetics | volume = 8 | issue = 10 | pages = 1955–63 | year = 1999 | pmid = 10469849 | doi = 10.1093/hmg/8.10.1955 | doi-access = free }}{{cite journal | vauthors = Hrabé de Angelis M, Balling R | title = Large scale ENU screens in the mouse: genetics meets genomics | journal = Mutation Research | volume = 400 | issue = 1–2 | pages = 25–32 | date = May 1998 | pmid = 9685575 | doi = 10.1016/s0027-5107(98)00061-x | bibcode = 1998MRFMM.400...25D }} Ethyl methanesulfonate (EMS) is also often used to generate animal, plant, and virus mutants.{{cite journal | vauthors = Flibotte S, Edgley ML, Chaudhry I, Taylor J, Neil SE, Rogula A, Zapf R, Hirst M, Butterfield Y, Jones SJ, Marra MA, Barstead RJ, Moerman DG | display-authors = 6 | title = Whole-genome profiling of mutagenesis in Caenorhabditis elegans | journal = Genetics | volume = 185 | issue = 2 | pages = 431–41 | date = June 2010 | pmid = 20439774 | pmc = 2881127 | doi = 10.1534/genetics.110.116616 }}{{cite book | vauthors = Bökel C | chapter = EMS Screens: From Mutagenesis to Screening and Mapping | title = Drosophila | series = Methods in Molecular Biology | volume = 420 | pages = 119–38 | year = 2008 | pmid = 18641944 | doi = 10.1007/978-1-59745-583-1_7 | isbn = 978-1-58829-817-1 }}{{cite journal | vauthors = Favor AH, Llanos CD, Youngblut MD, Bardales JA | title = Optimizing bacteriophage engineering through an accelerated evolution platform | journal = Scientific Reports | volume = 10 | year = 2020 | issue = 1 | page = 13981 | pmid = 32814789 | pmc = 7438504 | doi = 10.1038/s41598-020-70841-1 }}

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.{{cite web | vauthors = Krinke C | title = GMO directive : the origins of the mutagenesis exemption | work = Inf'OGM | date = March 2018 | url = https://www.infogm.org/6509 }}

{{main|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. 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.{{cite journal | vauthors = Shortle D, DiMaio D, Nathans D | title = Directed mutagenesis | journal = Annual Review of Genetics | volume = 15 | pages = 265–94 | year = 1981 | pmid = 6279018 | doi = 10.1146/annurev.ge.15.120181.001405 }} Such chemicals include aminopurine, which induces an AT to GC transition,{{cite journal | vauthors = Caras IW, MacInnes MA, Persing DH, Coffino P, Martin DW | title = Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells | journal = Molecular and Cellular Biology | volume = 2 | issue = 9 | pages = 1096–103 | date = September 1982 | pmid = 6983647 | pmc = 369902 | doi = 10.1128/mcb.2.9.1096 }} while nitrosoguanidine,{{cite journal | vauthors = McHugh GL, Miller CG | title = Isolation and characterization of proline peptidase mutants of Salmonella typhimurium | journal = Journal of Bacteriology | volume = 120 | issue = 1 | pages = 364–71 | date = October 1974 | doi = 10.1128/JB.120.1.364-371.1974 | pmid = 4607625 | pmc = 245771 }} bisulfite,{{cite journal | vauthors = Shortle D, Nathans D | title = Local mutagenesis: a method for generating viral mutants with base substitutions in preselected regions of the viral genome | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 75 | issue = 5 | pages = 2170–4 | date = May 1978 | pmid = 209457 | pmc = 392513 | doi = 10.1073/pnas.75.5.2170 | bibcode = 1978PNAS...75.2170S | doi-access = free }} and N⁴-hydroxycytidine may induce a GC to AT transition.{{cite journal | vauthors = Flavell RA, Sabo DL, Bandle EF, Weissmann C | title = Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 72 | issue = 1 | pages = 367–71 | date = January 1975 | pmid = 47176 | pmc = 432306 | doi = 10.1073/pnas.72.1.367 | bibcode = 1975PNAS...72..367F | doi-access = free }}{{cite journal | vauthors = Müller W, Weber H, Meyer F, Weissmann C | title = Site-directed mutagenesis in DNA: generation of point mutations in cloned beta globin complementary dna at the positions corresponding to amino acids 121 to 123 | journal = Journal of Molecular Biology | volume = 124 | issue = 2 | pages = 343–58 | date = September 1978 | pmid = 712841 | doi = 10.1016/0022-2836(78)90303-0 }} 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.

File:Site Directed Mutagenesis.png

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.

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.

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.

File:Site saturation mutagenesis.svg

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.{{cite journal |title=Analysis of Large-Scale Mutagenesis Data To Assess the Impact of Single Amino Acid Substitutions|author1=Vanessa E. Gray |author2=Ronald J. Hause |author3=Douglas M. Fowler|journal=Genetics |date= September 1, 2017 |volume= 207 |number= 1 |pages=53–61 |doi=10.1534/genetics.117.300064|pmid=28751422 |pmc=5586385 }} 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.{{cite journal|last=Reetz|first=M. T.|author2=Carballeira J. D. |year=2007|title=Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes|journal= Nature Protocols|volume= 2|issue= 4|pages=891–903|doi=10.1038/nprot.2007.72|pmid=17446890|s2cid=37361631}}{{cite journal |last1=Cerchione |first1=Derek |last2=Loveluck |first2=Katherine |last3=Tillotson |first3=Eric L. |last4=Harbinski |first4=Fred |last5=DaSilva |first5=Jen |last6=Kelley |first6=Chase P. |last7=Keston-Smith |first7=Elise |last8=Fernandez |first8=Cecilia A. |last9=Myer |first9=Vic E. |last10=Jayaram |first10=Hariharan |last11=Steinberg |first11=Barrett E. |last12=Xu |first12=Shuang-yong |title=SMOOT libraries and phage-induced directed evolution of Cas9 to engineer reduced off-target activity |journal=PLOS ONE |date=16 April 2020 |volume=15 |issue=4 |pages=e0231716 |doi=10.1371/journal.pone.0231716|pmid=32298334 |pmc=7161989 |bibcode=2020PLoSO..1531716C |doi-access=free }}

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.{{cite journal | vauthors = Parker AS, Griswold KE, Bailey-Kellogg C | title = Optimization of combinatorial mutagenesis | journal = Journal of Computational Biology | volume = 18 | issue = 11 | pages = 1743–56 | date = November 2011 | pmid = 21923411 | pmc = 5220575 | doi = 10.1089/cmb.2011.0152 | bibcode = 2011LNCS.6577..321P }} It provides a useful method to assess the combinatorial effect of a large number of mutations on protein function.{{cite journal | vauthors = Choi GC, Zhou P, Yuen CT, Chan BK, Xu F, Bao S, Chu HY, Thean D, Tan K, Wong KH, Zheng Z, Wong AS | title = Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9 | journal = Nature Methods | volume = 16 | issue = 8 | pages = 722–730 | date = August 2019 | pmid = 31308554 | doi = 10.1038/s41592-019-0473-0 | s2cid = 196811756 }} Large numbers of mutants may be screened for a particular characteristic by combinatorial analysis. In this technique, multiple positions or short sequences along a DNA strand may be exhaustively modified to obtain a comprehensive library of mutant proteins. 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.

{{further|Signature tagged mutagenesis|Transposon mutagenesis}}

The insertion of one or more base pairs, resulting in DNA mutations, is also known as insertional mutagenesis.{{cite journal | vauthors = Uren AG, Kool J, Berns A, van Lohuizen M | title = Retroviral insertional mutagenesis: past, present and future | journal = Oncogene | volume = 24 | issue = 52 | pages = 7656–72 | date = November 2005 | pmid = 16299527 | doi = 10.1038/sj.onc.1209043 | s2cid = 14441244 | doi-access = }} 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.{{cite book | vauthors = Vassiliou G, Rad R, Bradley A | title = Guide to Techniques in Mouse Development, Part B: Mouse Molecular Genetics, 2nd Edition | chapter = The use of DNA transposons for cancer gene discovery in mice | series = Methods in Enzymology | volume = 477 | pages = 91–106 | date = 2010-01-01 | pmid = 20699138 | doi = 10.1016/s0076-6879(10)77006-3 | publisher = Academic Press | isbn = 9780123848802 | edition = 2nd | veditors = Wassarman PM, Soriano PM }} Transposons, chromosomal segments that can undergo transposition, can be designed and applied to insertional mutagenesis as an instrument for cancer gene discovery. 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.

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.

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.{{cite web |url=http://www.bio.davidson.edu/courses/genomics/method/homolrecomb.html |title=Homologous Recombination Method (and Knockout Mouse) |work=Davidson College }}

{{main|CRISPR gene editing}}

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.{{cite journal |title=Seamless site-directed mutagenesis of the Saccharomyces cerevisiae genome using CRISPR-Cas9 |author1=Damien Biot-Pelletier |author2=Vincent J. J. Martin|journal=Journal of Biological Engineering|date=2016 |volume=10|page=6 |pmid=27134651 |pmc=4850645 |doi= 10.1186/s13036-016-0028-1 |doi-access=free }}{{cite journal |journal= J Genet Genomics|date=20 August 2015|volume=42|issue=8|pages=413–21| doi= 10.1016/j.jgg.2015.06.005|title=The application of CRISPR-Cas9 genome editing in Caenorhabditis elegans|author=Xu S| pmid=26336798 |pmc=4560834 }}

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.{{cite book |url=https://books.google.com/books?id=Yg3OBQAAQBAJ&pg=PA13 |title=Artificial DNA: Methods and Applications | veditors = Khudyakov YE, Fields HA |page=13 |publisher= CRC Press|date=25 September 2002|isbn=9781420040166 }}

• Genetic engineering
• Oncomouse
• Saturated mutagenesis
• Directed evolution

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Category:Genetically modified organisms

Category:Molecular biology techniques