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In 2012 a modified RAD tagging method called double digest RADseq (ddRADseq) was suggested. [10] [11] By adding a second restriction enzyme, replacing the random shearing, and a tight DNA size selection step it is possible to perform low-cost population genotyping.
The latter condition is of particular practical interest, since commercial restriction enzymes are usually supplied in a buffer containing a substantial amount of glycerol (50% v/v is typical), meaning insufficient dilution of the enzyme solution can cause star activity; this problem most often arises during double or multiple digests.
A restriction map is a map of known restriction sites within a sequence of DNA.Restriction mapping requires the use of restriction enzymes.In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA.
Different restriction enzymes acting on different recognition sites produce different DNA fragments. The term restriction enzyme originated from the studies of phage λ, a virus that infects bacteria, and the phenomenon of host-controlled restriction and modification of such bacterial phage or bacteriophage. [12]
HaeIII along with other restriction enzymes were discovered in 1970 by Werner Arber and Matthew Meselson. The HaeIII methyltransferase also known as MTase gene from Haemophilus aegyptius (recognition sequence: 5′-GGCC-3′) was made into Escherichia coli (E.coli) in the plasmid vector pBR322.
Restriction digest is most commonly used as part of the process of the molecular cloning of DNA fragment into a vector (such as a cloning vector or an expression vector).The vector typically contains a multiple cloning site where many restriction site may be found, and a foreign piece of DNA may be inserted into the vector by first cutting the restriction sites in the vector as well the DNA ...
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BglII catalyses phosphodiester bond cleavage at the DNA backbone through a phosphoryl transfer to water. [1] Studies on the mechanism of restriction enzymes have revealed several general features that seem to be true in almost all cases, although the actual mechanism for each enzyme is most likely some variation of this general mechanism.