Mutagenesis

Mutagenesis ( a composite, see also Genesis or Genesis ) is the generation of mutations in the genetic material of living organisms. In the biological and medical research as well as in the culture, mutagenesis is used to achieve desired properties.

The other hand, indicates the mutagenicity level of the ability of a condition ( substance or radiation) to mutagenesis.

Conventional mutagenesis

In conventional mutagenesis, the genetic makeup of an organism is not specifically changed. For this to be mutagenic breeding organisms, ie mutagenic conditions exposed. These range from the radiation (eg, UV light ) to the use of chemical substances with defined mutagenicity. It is impossible to predict exactly where it comes in the genome to a mutation. Instead of the desired organism through a screening process is sought, for example, only the mutant bacterial colonies that grow on a particular medium are grown. Another method is the random mutagenesis using a transposon. This transposon should be located on a plasmid, wherein the associated transposase should be outside of the coding region of the transposon. Is used for this method so-called Suizidplasmide which have a non-functional OriV to prevent replication of the plasmid. After the transformation of the organism of interest, the transposition is made at a random gene region on the bacterial chromosome. Since here the so-called composite transposons such as Tn5 are used, the transpose after the cut-and -paste mechanism, the plasmid is then interrupted and will be removed in the cell by restriction. Once inserted the transposon remains ideally in the region of the chromosome. Used this method for the detection of genes for a given phenotype. By screening for the colonies with mutations in this gene this, the exact position on the chromosome can be detected.

Site-specific mutagenesis

When site-specific or site-directed mutagenesis ( Site- directed mutagenesis ) is using the recombinant DNA allows selective modification of DNA. It can therefore efficiently exchanged individual nucleic bases of a gene or even entire genes are removed. This process is now a widely used method in molecular biology, which ranges from the change of a gene on a plasmid up to the knock-out mouse.

Methods

Over time, a variety of methods have been developed. These include the cassette mutagenesis, primer extension mutagenesis, ligation - during- amplification ( QuikChange ), megaprimer mutagenesis and overlap extension PCR. All methods is the use of at least one synthetic, a mutation -containing oligonucleotide and the use of DNA to be mutated as a template, usually a plasmid in common.

Because the yield is not targeted to the mutation at 100 %, a selection is necessary. In this case, the entire reaction product obtained DNA is introduced into host cells, typically E. coli, then screened for colonies which selectively include DNA with the desired mutation (e.g. by transformation ), and. For a simplified screening, it may be useful to introduce or remove restriction enzyme sites on the mutagenic oligonucleotide.

History

An important milestone in modern molecular biology in 1978 was the initial characterization of site-directed mutagenesis using oligonucleotides for the in vitro synthesis of mutated DNA. For the establishment of this technique Michael Smith received the 1993 Nobel Prize in Chemistry.

Random mutagenesis

The random mutagenesis ( engl. random mutagenesis ) embodies virtually the opposite of site-specific mutagenesis. The aim of this process is the more or less random substitution of nucleotides of a DNA molecule for then a thus obtained pool of clones with various mutations to isolate those with the desired properties and to be identified by subsequent DNA sequencing. Random mutagenesis is usually based on the use of error-prone DNA polymerases. The mutagenesis can be used in addition, controlled with the nucleoside triphosphate concentration, the use of nucleotide analogues or the addition of Mn2 . Alternatively, a site-directed mutagenesis using random mutations or nucleotide analogues containing entertaining oligonucleotides to generate random mutations at defined sites of the DNA template to be used.