Restriction enzymes - The most useful tool in Molecular biology
The restriction enzymes/endonucleases (REases) were first discovered by Swiss microbiologist Werner Arber (with Stuart Linn) in 1978 (for which they received Nobel prize) during the study of host controlled restriction of bacteriophases (from E. coli strain). Now it has become the most essential tool in genetic engineering & molecular biology that can cleave the sugar phosphate backbone of the DNA at specific nucleotide sequence (called recognition sequence which is about 4-6 nt long). As example, restriction enzyme HaeII from the bacterium Hemophilus aegypticus cuts the DNA only at particular sequence:
5’ GGCC 3’
3’ CCGG 5’
The recognition sequence length dictates the cutting frequency of the enzyme in a random DNA sequence. Such as enzymes with 6 bp long recognition site will cut the DNA in every 46(i.e. 4096) bp. The recognition site of one RE can be similar with another RE and such REs are called isoschizomers such as SacI and SstI [1]. REs are evolved from bacteria and named by the host, for example, EcoR1 are derived from E. coli strain RY13.
Restriction enzymes are mainly of 4 types on the basis of subunit composition, sequence specificity, cleavage position and cofactor requirements [2].Type I restriction enzymes cleave DNA at random sites that are far from its recognition sequence. The most common (93% of REs) commercially available restriction enzyme is Type II restriction enzyme that cleaves DNA in the presence of Mg2+ within its recognition sequence and it does not require ATP hydrolysis such as HindIII [3]. Type II restriction enzymes are of several types, such as Type IIG (that cleaves outside its recognition sequence such as Acul), type IIP (that cleaves symmetric sequences) and type IIS (that cleaves asymmetric sequences such as FolkI). TypeIII restriction enzyme cleaves outside their recognition sequences and require two sequences with opposite orientation within the same DNA molecule. Type IV restriction enzymes cleave only modified/methylated DNA.
Restriction digestion of DNA double helix results in highly reactive sticky ends or cohesive ends that can be bound by DNA ligase. All restriction enzymes are heat sensitive. Their activity is highly affected by enzyme concentration, sequence context, incubation temperature and buffer composition. Deviation of any of these factors results in ineffective restriction digestion at cognate restriction site or cleavage at non-cognate star sites (Star activity) [4]. Some factors can induce star activity in restriction digestion such as high pH, prolonged reaction time, presence of organic solvents (e.g. DMSO) and glycerol concentration of the reaction mix. Recently New England Biolabs developed High Fidelity engineered restriction endonucleases that have reduced star activity and rapid digestion [5].
Applications:
- Cloning utilizes restriction enzymes along with DNA ligase for the study and production of recombinant proteins.
- DNA mapping or restriction mapping for the detection of single nucleotide polymorphism and mutation to identify genetic disorder loci
- Epigenetic modification study
- Synthetic biology utilizes restriction enzymes to create novel technologies such as Bio-BrickTM and Golden Gate assembly.
- DNA Library construction by SAGE (Serial Analysis of Gene Expression) to identify mutation in cancer research [6]
- Gene editing, DNA sequencing, DNA fingerprinting and in vast area of recombinant DNA and biotechnology
References:
