Restriction digestion is an enzymatic reaction, by DNA molecule

Restriction digestion is an enzymatic reaction, by DNA molecule: 

           As restriction digestion is an enzymatic reaction, it depends
on the reaction conditions such as restriction time provided, p1-I,
temperature, ionic strength of the reaction buffer etc. Commercially
available restriction enzymes are provided in Units (U), rather
than in volumetric units. One unit is defined as the quantity of
enzyme required to digest I ig of DNA in one hour. As all the
type II restriction enzymes require Mg2 for their activity, the
reaction mixture therefore must contain Mg in the form of
magnesium salt (MgC12). Each enzyme has specific requirements
of the above conditions, although most of them function at neutral
pH and ambient temperature (37°C). To avoid sudden changes in
pH, the digestion is performed in a buffer, preferably in Tris-HCL
with NaCI and MgCI,. By manipulating the reaction condition
the activity of the restriction enzyme may be regulated. A typical
example of this kind is star activity, where the enzyme recognizes
a different sequence or part of recognition sequence under
suboptirnal salt concentration. The enzyme EcoRI at low saLt
concentration recognizes internal 5’AATT3’ instead of the normal
5’GAATTC3’. Depending of the objective, restriction enzymes may
be used for complete or partial digestion. Besides, combinations
of restriction enzymes may also be used to generate fragments
with different terminal sequences. Relative positions of different
restriction sequences of a DNA fragment, clone, plasmid or
chromosomes generated through digestion with different
restriction endonucleases can be used as a restriction map. Such
maps are very useful in molecular cloning and genetic engineering
as they reveal the eact positions of restriction sites thereby
helping to choose the restriction enzyme for digestion.

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