Restriction enzyme

FACTORS AFFECTING THE ACTIVITY OF RESTRICTION ENZYMES
Depending on the substrate DNA and the reaction conditions, restriction enzymes show a wide variation of cleavage and possible star activity. In order to obtain the desired cleavage, it becomes important to control the following factors:

1. 
   Star activity: Under sub-optimal reaction conditions, some restriction enzymes cleave base sequences at sites different from the defined recognition sequence. In other words, they cleave at non-specific sites. This phenomenon is called star activity. Some of the factors that induce star activity are high salt and glycerol concentration, presence of impurities, excessive enzyme compared to substrate DNA, increased incubation time, or incompatible buffer and cofactor.
   
2. 
   Methylated DNA: Several DNA molecules are methylated at the recognition site, making them resistant to cleavage by certain restriction enzymes. For example, most E. coli strains express Dam or Dcm methyltransferases that methylate specific recognition sites to form G6mATC and C5mCA/TGG, respectively. G6mATC is resistant to cleavage by Mbo I.
   
3. 
   Temperature: Most endonucleases optimally digest the target DNA at 37 °C. However, there are some exceptions with lower or higher optimal temperatures. For example, Taq I optimally digests at 65 °C and Apa I (Catalog No. 10899208001) digests at 25 °C.
   

ISOSCHIZOMERS AND NEOSCHIZOMERS⁶
Isoschizomers are restriction enzymes with the same recognition sequence and cleavage sites. Example: Sph I (CGTAC/G) and Bbu I (CGTAC/G)

Neoschizomers are restriction enzymes with the same recognition sequence but cleave the DNA at a different site within that sequence. Example: Tai I (ACGT/) and Mae II (A/CGT)

PRODUCTS OF RESTRICTION DIGESTION
Restriction digestion of double-stranded DNA produces two kinds of ends: Sticky ends and Blunt ends.
Blunt ends possess a 5’-phosphate group that promotes ligation. They are universally compatible with other blunt-ended DNA.
Blunt ends generated by EcoR V

Sticky ends are small stretches of single-stranded DNA capable of self-ligation or ligation with a complementary region from another DNA molecule. The sticky ends possess 3’- or 5’-overhangs of 1–4 nucleotides.
5’ Cohesive end generated by Bln I (Catalog No. 11558170001)

3’ Cohesive end generated by Kpn I

BUFFER SYSTEM
Our restriction enzyme collection has been optimized for digestion using five unique buffers. When digesting DNA using a single enzyme, use the buffer supplied with the enzyme. For double digestion of DNA, use the buffer in which both the enzymes show 100% activity. Alternatively, the optimal buffer can be determined from the chart of common double digestions. In some cases, sequential digestion is recommended due to buffer incompatibility (composition or temperature). For protocols on restriction digestion with single enzyme, two restriction enzymes and sequential DNA Digestion, refer to Restriction Enzyme Digest Protocol. The selection of the correct buffer is crucial for obtaining high activity of both enzymes and for avoiding star activity. The choice of five buffers allows the user to select compatible buffers for the desired digestion. Most digestions require 1–2 buffers, which becomes cost-effective.
APPLICATIONS
The ability of restriction endonucleases to cleave DNA at specific recognition sites has enabled extensive use of these enzymes as essential tools in several molecular biology techniques. Some of the major applications are explained below:

1.