Restriction digest

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A restriction digest is a method of cutting DNA at specific DNA sequences using restriction enzymes . The restriction digest is used to characterize DNA on the basis of the characteristic DNA fragments formed ( restriction analysis ) and to prepare DNA for cloning .

properties

During restriction digestion, DNA is incubated with the restriction enzyme in a buffer solution suitable for the restriction enzyme used . In the case of unsuitable buffer compositions, star activity of the restriction enzyme can arise. The restriction enzymes each have a DNA sequence (mostly double-stranded) to which they bind ( restriction site ). The included restriction enzyme endonuclease results in a sequence-specific hydrolysis of DNA. Some restriction enzymes produce blunt ends , while others produce sticky ends .

Cloning

DNA with sticky ends lead to a higher yield in a subsequent ligation in the course of a cloning. Usually two different interfaces are used. The orientation of the DNA to be inserted can be controlled by choosing two different sticky-ends-generating restriction enzymes during a cloning, while with blunt ends 50% of the products of a ligation have the wrong orientation of the inserted DNA to the promoter and therefore no gene expression can take place. Both the vector (mostly a plasmid ) and the DNA sequence to be inserted must then have both interfaces and both must also enable the correct orientation of the start codon of the DNA sequence to be inserted relative to the promoter.

There is often a multiple cloning site in the vector , which consists of a series of different restriction sites. This allows different restriction enzymes to be used.

If the DNA sequence to be inserted does not yet contain any suitable restriction sites, these can be generated via a polymerase chain reaction with primers which have each been extended by the corresponding cleavage site. Restriction enzymes require a number of nucleotides between the start of the primer and the recognition sequence of the respective restriction enzyme of up to six nucleotides.

Restriction analysis

Restriction analysis is a comparatively simple and inexpensive method for determining the identity of DNA with a known sequence or for comparing the identity of two DNA samples without the sequence having to be known. For this purpose, the DNA is treated with restriction enzymes and then separated by agarose gel electrophoresis . By estimating the size of the resulting DNA fragments in comparison to a DNA ladder, it is possible to check what can be expected from an in silico analysis of the known DNA sequence ( restriction map ) in the event of a restriction with a specific restriction enzyme. Further developments in restriction analysis are e.g. B. the RFLP , the ARDRA and the TRLP .

literature