Molecular Biology. Practical Guide - Velikov V.A. 2013

DNA Restriction

Restriction Endonucleases (restriction Enzymes) are widely used as "molecular scissors" in Genome Mapping, Gene cloning, genotyping, and other molecular genetic Applications. Restriction enzymes cleave DNA molecules at specific nucleotide sequences known as recognition sites. Unlike exonucleases, which degrade DNA progressively from the ends, these site-specific endonucleases make internal cuts within the DNA strand. The first restriction enzymes were isolated by Smith (Smith, 1970; Smith, Nathans, 1973), while The phenomenon of DNA Restriction-modification itself was first discovered by Luria (Luria, 1952) while studying phage propagation across different bacterial strains.

Type II restriction enzymes recognize palindromic nucleotide sequences—sequences with a twofold axis of Symmetry. For instance, the six-nucleotide recognition site of the EcoRI restriction enzyme, -GAATTC-, reads identically on the complementary DNA strand in the same 5’→3’ direction. The Cleavage site is located between G and the first A, where the phosphodiester bond is hydrolyzed. The resulting 5’-overhanging "sticky end" generated by restriction is 4 NUCLEOTIDES long:

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These enzymes typically function as dimers, with each protein subunit hydrolyzing One DNA strand independently of the other. However, they act significantly slower on single-stranded DNA. Depending on the relative position of the cuts on the complementary DNA strands, the resulting restriction fragments feature either 5’-overhangs (EcoRI, BamHI, HindIII) or 3’-overhangs (PstI) sticky ends. The majority of restriction enzymes yield 5’-overhanging sticky ends (see App. 3).

Enzyme activity and the completeness of DNA Digestion can be influenced by a variety of factors, including incubation Temperature, the ionic COMPOSITION OF THE buffer, the DNA extraction method (and consequently the degree of contamination), as well as the level and Specificity of DNA Methylation. The results of restriction digestion are evaluated using agarose gel DNA Electrophoresis.

Enzymatic digestion of small plasmid or Viral DNA molecules with a given restriction enzyme produces a strictly defined, limited number of restriction fragments (App. 3, 13). Due to the vast number of restriction sites and inevitable DNA fragmentation during isolation from Cells, genomic DNA preparations yield a continuous smear of DNA fragments upon electrophoresis (App. 13).

The DNA Restriction-modification system is a bacterial enzymatic defense system comprising two Proteins—a restriction enzyme and a methylase—both specific to the same recognition site. This system destroys foreign DNA entering The Cell. Its primary function is to protect the cell against invading foreign genetic material, most notably Bacteriophages and Plasmids. The bacterial genome is protected from its own restriction enzyme through the methylation of adenine or cytosine by the corresponding methylase enzyme: methylated sites cannot be cleaved by the restriction enzyme.

For any DNA with a known Primary Structure, the number and Location of all restriction sites can be determined using specialized restriction mapping software, such as RestrictionMapper (http: //www. restrictionmapper. org).