For cloning of DNA, often we need to cut the DNA at specific sites, which are recognized and cleaved by specific enzymes (endonucleases, which cleave DNA at interval sites), described as
restriction enzymes. These restriction enzymes recognize short sequences of double stranded DNA as targets for cleavage. Different enzymes recognize different, but specific sequences, each ranging in length from 4 to 8 base pairs. Each enzyme is named by a three letter (or four letter) abbreviation (letters are italicized) that identifies its origin. Roman numerals (I, II, III
, etc.) are added to distinguish several enzymes with same origin. For instance
EcoRIis derived from
E. coli and
HpaIis derived from
Haemophilus parainfluenzae (Table 39.1). By locating the positions of the cleavage sites of a number of restriction enzymes in a DNA segment, restriction maps can be prepared (consult next main topic).
Besides cleavage, modification in the form of methylation is also brought about by some enzymes called modification enzymes (sometimes also called
methylases). This methylation distinguishes genes in different states of functioning. There are alsp enzymes which perform the function of restriction (cleavage) and modification (methylation). Based on these attributes restriction enzymes have been grouped into two classes :
(a) type II restriction enzyme systems (e.g. ZscoRI), which have separate enzymes for modification and restriction and (b)
type I (
EcoK and
EcoB)and type
III (
EcoT1
, Eco15)enzyme systems, in which same enzyme possesses both activities (bifunctional), although the restriction and modification sites differ in position. (For more details, consult
Chemistry of the Gene 2. Synthesis, Modification and Repair of DNA).
Of the above two classes of restriction enzymes, type II enzymes are most important for cloning purposes. Some enzymes introduce staggered cuts, others generate blunt ends. Enzymes with 4bp target sites are used when frequent cuts are desired and those with 8bp are used when rare cuts are desired to get long DNA segments. Otherwise majority of enzymes used have 6 bp target sites. Some of them can cleave both methylated as well as unmethylated targets, but majority of them cleave only unmethylated targets. Details of some of these enzymes are listed in Table 39.1.
