Prokaryotic and eukaryotic cells are constantly exposed to extrinsic and intrinsic stimuli that cause DNA damage, which can lead to mutations if left unrepaired.Lombard et al. 2005;Boesch et al. 2011). To survive under these constant genotoxic conditions and still faithfully replicate the genome, prokaryotic and eukaryotic cells have evolved specific DNA repair machinery that repairs DNA damage and slows the accumulation of genetic mutations.Lombard et al. 2005;Boesch et al. 2011). However, not all mutations affect the biological function of a protein. Once a mutation has been identified in an organism, functional characterization is still required to understand the effect of the mutation on the function of a specific protein or non-coding RNA.
Several mutagenesis methodologies have been developed to introduce mutations at predetermined sites or regions in mammalian genes (seebox 1). Below we describe the three main types of mutagenesis approaches and provide a list of detailed protocols for commonly used methods (seetabla 1). These nine different methods of performing mutagenesis can be used for the introduction of mutations according to the requirements of the researcher. The terminology for mutagenesis is defined inbox 2.
Table 1.
HISTORICAL BACKGROUND
The road to the development of modern in vitro mutagenesis methods is paved with Nobel laureates as progress has moved from the level of treating whole organisms to targeting specific sites on the DNA molecule. Hermann J. Muller (1890-1967), then affiliated with the University of Texas, moved the scientific world in 1926 when he described evidence for genetic mutations and chromosomal changes inDrosophilaproduced by X-rays. He announced his results at a scientific meeting in 1926 and later published them in the article "Artificial Transmutation of the Gene" inScience(Müller 1927). In the experiments, the genesis of mutations by X-rays played an important roleneurosporapublished in 1941 by George Beadle and Edward Tatum proposing their one gene, one protein hypothesis (Janitor and Tatum 1941).
H.J. Muller spent some time at the University of Edinburgh from 1938 to 1940, where he worked with Charlotte Auerbach (1899-1994) on substances that could cause mutations. with J.M. Robson in the early 1940s, Auerbach used mustard gas to induce mutationsDrosophila(Auerbach en Robson 1947;Stevens et al. 1950). In a later message, Auerbach related that:
It seems to me that there could be many ways to influence the chemical specificity of the gene or the physical integrity of the chromosomes: direct chemical reaction with proteins or nucleic acids; energy release near a chromosome; inactivation of enzymes related to chromosome metabolism; interference with gene duplication by competitive analogs, etc.Auerbach 1951).
The questions raised by Auerbach were answered after the determination of the structure of DNA in 1953 by James D. Watson and Francis H.C. Crick: a discovery that changed the nature of mutagenesis studies. Mutagenesis techniques became highly specific as a result of Michael Smith's (1932-2000) research on oligonucleotide synthesis and his 1975 sabbatical in Fred Sanger's sequencing lab.Escherichia coliphage ϕX174. Smith realized that a mutagenic method was needed to attack specific bases. Their studies showed that small oligonucleotides could form stable duplexes at low temperatures, while work by Clyde A. Hutchison III and Marshall H. Edgell showed that “point mutations could be reversed by hybridizing mutant phage ϕX174 to wild-type antisense fragments.” DNA before transfection. Using ϕX174 DNA as a template, a 120 nt oligomer with a single nucleotide mismatch as a primer, andE coliDNA polymerase produced a closed circular double-stranded DNA with the oligonucleotide incorporated into one strand (Hutchison et al. 1978). Almost at the same time, the first specific restriction endonucleases were discovered, which made it possible to isolate specific DNA fragments.Smith en Wilcox 1970).
A final key element emerged in 1983 when Kary Mullis invented the idea of the polymerase chain reaction (Saiki et al. 1985;Mullis et al. 1986). Polymerase chain reaction (PCR) quickly became an integral part of in vitro site-directed mutagenesis techniques.
As for the number of Nobel Prizes, Müller's (Physiology or Medicine) was awarded in 1946, and Smith's in 1993 (Chemistry) was shared with Mullis. Beadle and Tatum were cited in 1958 (Physiology or Medicine), and Watson and Crick in 1962 (Physiology or Medicine).
Table 2.
MUTAGENIC TERMINOLOGY
There is a large amount of mutagenesis terminology, some of it confusing, with different names for the same approach. The following list clarifies the terminology of the multitude of mutagenesis approaches.
5' Complementary Mutagenesis
A PCR-based method useful for adding a new sequence or chemical group to the 5' end of a PCR product.
Alanine-scanningmutagenese
Method used to determine the structure-function relationship of a particular protein. Alanine is the substitution residue of choice because it removes the side chain beyond the β-carbon and yet does not change the conformation of the main chain or impose extreme electrostatic or steric effects. (See also Scanning mutagenesis.)
cassette-mutagenese
It is used for the efficient insertion of mutagenic oligodeoxynucleotide cassettes, which allow saturation of a target amino acid codon with multiple mutations.
chemical mutagenesis
It uses the nature of chemical mutagens, such as hydroxylamine andNorth-ethyl-North-nitrosourea, to introduce random mutations into the target DNA sequence.
circular mutagenesis
It introduces site-directed mutations in circular DNA molecules of interest via mutagenic primer pairs.
Mutagenese van codoncassettes
It is based on the use of universal mutagenic cassettes to deposit individual codons at specific sites on double-stranded DNA.
deletion mutagenesis
It is used to produce randomly placed, targeted deletions in large sections of DNA to construct deletion mutants.
directed evolution
It is used in protein engineering to harness the power of natural selection to develop proteins or RNAs with desirable properties not found in nature.
dishes mutagenesis
See site-directed mutagenesis.
Domain mutagenesis
It is used to introduce multiple mutations into a defined region of cloned DNA.
insertionele mutagenese
It is used to mutagenize a DNA template by inserting one or more bases. These insertions can occur naturally, such as via transposons, or artificially in a laboratory.
Linker-scanningmutagenese
In linker scanning mutagenesis, a library of 5' and 3' deletants is first created and the ends are ligated to a linker oligonucleotide. Depending on the DNA sequence of the individual removers, paired combinations are chosen and used to create a new DNA fragment in which the linker sequence accurately replaces part of the original sequence without changing the spacing of the surrounding nucleotides.
PCR-based mutagenesis with megaprimer
Two outer oligonucleotide primers and one inner mutagenic primer are used in two rounds of PCR to mutagenize a DNA sequence. The first round of PCR is performed using one of the outer primers and the mutagenic primer containing the desired mutation. This generates a PCR intermediate which is then used as a "megaprimer" for the second round of PCR, along with the other outer primer. The final PCR product is cloned into appropriate vectors and used in subsequent applications.
malincorporative and mutagenic
It uses reverse transcriptases, such as error-prone polymerases, to create mutations.
Mismatch-mutagenese
Create DNA-based pair-specific mismatches.
Multi-site directed mutagenesis
It allows mutagenesis at multiple sites simultaneously with a single oligonucleotide primer per site.
Oligonucleotide-directed mutagenesis
It uses a mutagenic oligonucleotide primer to introduce a mutation into a DNA strand. It may or may not be accompanied by PCR.
PCR-mutagenese
Any technique that uses PCR to generate a mutation in a specific DNA sequence.
PCR site-directed mutagenesis
It uses mutagenic oligonucleotide primers to introduce the desired mutations.
random mutagenesis
For example, random mutations in a specific DNA sequence are caused by UV radiation.
Random scanning mutagenesis
An oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein.
Retroviral insertional mutagenesis
Retrovirus particles are used to create insertional mutagenesis.
Saturation mutagenesis
A form of site-directed mutagenesis, which attempts to generate all possible mutations (or as close as possible) to a specific site or narrow region of a gene.
The mutagenesis scan
It is used to understand structure-function relationships by creating a library of mutants with an alanine or cysteine residue at any position in the protein. (See also alanine scanning mutagenesis).
Sequence Saturation Mutagenesis
A method of creating mutations at any nucleotide position in a particular target sequence.
Mutagenesis with signature tag (STM)
Genetic engineering used to study the function of genes. STM can be used to infer the function of a particular gene by looking at the effects of mutations on the phenotype. The original and most common use of STM is to discover which genes of a pathogen are involved in the virulence of its host so that better medical treatments can be designed.
Site-directed mutagenesis
Also called "site-specific mutagenesis" or "oligonucleotide-directed mutagenesis". This is a technique where a desired mutation is created at a defined site on a DNA molecule.
Site-specific mutagenesis
See site-directed mutagenesis.
Transposon-mutagenese
Also called "transposition mutagenesis," this is a biological process that allows genes to be transferred to a host organism's chromosome, disrupting or altering the function of an existing gene.