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.