Mutagenesis
The mutagenesis (a compound , see also Genesis or Genesis ) is the generation of mutations in the genetic material of living things . Mutagenesis is used in biological and medical research as well as in breeding in order to achieve desired properties.
The mutagenicity other hand, refers to the degree of the ability of a condition (substance or radiation) for mutagenesis.
Conventional mutagenesis
In conventional mutagenesis, the genome of a living being is not specifically changed. For this purpose, the organisms to be grown are mutagenic , i. H. mutagenic conditions. These range from irradiation (e.g. with UV light) to the use of chemical substances with a defined mutagenicity. It is impossible to predict exactly where in the genome a mutation will occur. Instead, the desired organism is searched for via a screening method, e.g. B. only the mutated bacterial colonies that grow on a particular medium are grown.
Another method of random mutagenesis is mutagenesis using a transposon . This transposon should be on a plasmid , the associated transposase should be outside the coding region of the transposon. So-called suicide plasmids , which have a non-functional OriV , are used for this method in order to prevent replication of the plasmid. After the transformation of the organism of interest transposition occurs at a random gene region on the bacterial chromosome . Since so-called composite transposons such as Tn5 are used here, which transpose according to the cut-and-paste mechanism , the plasmid is then interrupted and is removed in the cell by restriction. Once inserted, the transposon ideally remains in the respective region of the chromosome. This method is used to detect genes for a specific phenotype . By screening for colonies with mutations in this sought-after gene, the exact position on the chromosome can be determined.
Site-specific mutagenesis
In the site-specific (also: site-directed ) or site-directed mutagenesis (English site-directed mutagenesis ), with the aid of recombinant DNA techniques brought about a targeted change in the DNA. Individual nucleobases of a gene can be exchanged in a targeted manner or entire genes or gene segments can be removed. This method is now a widely used method in molecular biology , which has a wide range of applications, from changing a gene on a plasmid to generating transgenic organisms such as a knockout mouse .
In recent years, new methods of targeted mutagenesis have been developed, which are summarized under the term genome editing .
Methods
A variety of methods have been developed over time. These include a. the cassette mutagenesis , primer extension mutagenesis , ligation-during-amplification (QuikChange), Mega primer mutagenesis and the overlap extension PCR . All the methods have in common the use of at least one synthetic oligonucleotide containing a mutation and the use of a DNA to be mutated as a template, usually a plasmid. A mutagenesis with random mutations in a certain area is saturation mutagenesis .
Prior to the transformation of the mutant DNA in bacteria one non-mutated parental DNA can destroy. The restriction enzyme Dpn I is added to this. It cuts and destroys DNA that comes from organisms because adenine is methylated in the statistically frequent DNA sequence GATC. The mutated DNA generated by PCR remains because it is unmethylated on the adenine.
Since the yield of targeted mutated DNA is still not 100%, a selection is necessary. Here, all of the DNA obtained as a reaction product is introduced into host cells, usually E. coli (e.g. by transformation) and then screened for colonies which selectively contain DNA with the desired mutation. To facilitate screening, it can be useful to insert or remove interfaces for restriction enzymes on the mutagenic oligonucleotide. The success of the targeted mutagenesis is usually checked by DNA sequence analysis .
history
An important milestone in modern molecular biology was the first description of site-specific mutagenesis with the aid of oligonucleotides for the in vitro synthesis of mutated DNA in 1978 . Michael Smith received the Nobel Prize in Chemistry in 1993 for establishing this technology .
Random mutagenesis
The random mutagenesis (English random mutagenesis ) embodies practically the opposite of the site-specific mutagenesis. The aim of this process is the more or less random exchange of nucleotides of a DNA molecule in order to then isolate the pool of clones with different mutations obtained in this way with the desired properties and to identify them by subsequent DNA sequencing . Random mutagenesis is usually based on the use of error-prone DNA polymerases . The rate of mutagenesis can also be controlled via the nucleoside triphosphate concentration used, the use of nucleotide analogs or an addition of Mn 2+ . Alternatively, a site-specific mutagenesis with the aid of random mutations or oligonucleotides containing nucleotide analogs can be used to generate random mutations at defined positions in the DNA template.
Individual evidence
- ↑ Ling, MM & Robinson, BH (1997): Approaches to DNA mutagenesis: an overview. In: Anal. Biochem. Vol. 254, pp. 157-178. PMID 9417773
- ↑ ´´ restriction enzyme DpnI. Retrieved September 24, 2014 ´´
- ↑ DNA methylation in bacteria
- ^ Turchin, A. & Lawler. JF (1999): The primer generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis. In: Biotechniques. Vol. 26, pp. 672-676. PMID 10343904
- ↑ Hutchison, CA et al. (1978): Mutagenesis at a specific position in a DNA sequence. In: J. Biol. Chem. Vol. 253, pp. 6551-6560. PMID 681366 PDF
- ↑ Information from the Nobel Foundation on the 1993 award ceremony to Michael Smith (English) , Nobel Lecture (PDF; 802 kB)
See also
Web links
- Laborjournal: Targeted Mutagenesis, Part I ( Memento of March 16, 2009 in the Internet Archive ), Part II ( Memento of December 12, 2008 in the Internet Archive ), Part III ( Memento of March 18, 2009 in the Internet Archive )