Restriction enzyme
Restriction enzymes cleave the sugar-phosphate backbone of double-stranded DNA. They recognize a specific site of double-stranded DNA and cleave it within or adjacent to their recognition site. The optimal activity of each restriction enzyme is affected by conditions such temperature, pH, enzyme cofactor, salt composition, and ionic strength.
Reaction conditions
Incubation temperature affects the activity of restriction enzyme greatly. In general, the incubation time reflects the growth temperature of the bacterial strain from which it originates. Most of the restriction enzymes have an optimum temperature of 37°C and an optimum pH of pH 7.2 or pH 8.5. These physical environment requirements must be met to ensure that the enzyme reaches its maximum activity. Outside the desirable physical condition range, the enzyme can denature. Enzymes work very specifically; therefore, they will not work properly if their structure deteriorates. Because each restriction enzyme is isolated from different bacteria and have different optimum reaction condition, they require specific buffer that is used for each restriction reaction.
Types of restriction enzymes
Restriction enzymes can be divided into 4 groups based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements. The essential difference between them are summarized in Table 1.
Table 1. Types of restriction enzyme
| Types | Characteristics |
|---|---|
| Type I | Type I restriction enzyme system compromises of one enzyme with different subunits for recognition, cleavage, and methylation. This enzyme recognizes and methylates at the same sequence but cleaves DNA up to 1000 base pairs away from the initial recognition site. |
| Type II | Type II restriction enzyme compromises two different enzymes, which cleave or modify the recognition sequence. |
| Type III | Type III restriction enzyme system consists of one enzyme with two different subunits, each for recognition and modification or cleavage. This enzyme recognizes and methylates at the same sequence but cleaves 24-26 base pairs away from the initial recognition site. |
| Type IIs | Type IIs restriction enzyme system compromises two different enzymes with asymmetrical recognition sequence. Cleavage occurs on one side of recognition site up to 20 base pairs away. |
The most useful type of restriction enzymes that have been sold commercially is type II restriction enzyme. Because it consists of two different enzymes, it is possible to cleave DNA without modification. Moreover, the type II restriction enzyme activity does not require a cofactor such as ATP or S-adenosylmethionine.
Restriction enzyme recognition sequence
Most type II restriction enzymes recognize and cleave DNA within a specific sequence of 2-8 nucleotides, which is palindromic. A palindromic sequence means that the nucleotide base sequence reads the same backwards (3' to 5' direction) and forwards (5' to 3' direction). Type II restriction enzymes produce three different ends:
a) Blunt-end
This type of enzyme cuts precisely at the same site in both DNA strands generating blunt end DNA fragments without overhangs.
b) 5' sticky end
This type of enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends.
c) 3' sticky end
This type of enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 3' ends.
Table 2. Restriction enzyme recognition
| Enzyme | Recognition sequence | Ends |
|---|---|---|
| HindIII | A/AGCTT | 5'sticky ends |
| BglI | GCCNNNN/NGGC | 3'sticky ends |
| EcoRI | G/AATTC | 5'sticky ends |
| BamHI | G/GATCC | 5'sticky ends |
| EcoRV | GAT/ATC | blunt ends |
| SalI | G/TCGAC | 5'sticky ends |
| FaeI | CATG/ | 3'sticky ends |
| KpnI | GGTAC/C | 3'sticky ends |
| XhoI | C/TCGAG | 5'sticky ends |
| DpnI | GA/TC | blunt ends |
| SmaI | CCC/GGG | blunt ends |
| XbaI | T/CTAGA | 5'sticky ends |