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Genial -. Worksheet IX.5. Restriction enzymes. Types of Restriction/Modification Systems

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  1. Genial - Worksheet IX.5 Restriction enzymes • Types of Restriction/Modification Systems • Restriction/Modification (or restriction/methylation) enzyme systems have been subdivided into three categories: Type I, type II and type III. The major differences are that type II enzymes work separately and independently from the functionally pertinent methylation enzymes, while type I and type III enzymes carry both restriction and methylation properties in one enzyme, consisting of two or three heterologous subunits. Type I and type III restriction/modification enzyme systems are less well known and less common. Type I enzymes cut the DNA randomly far from the recognition site. Type III enzymes cut the DNA 20-25 nucleotides up- or down-stream the recognition site. • Type II systems produce the well-known restriction enzymes available from commercial suppliers and are the most useful in molecular biology applications. Both the restriction and methylation enyzmes have an absolute requirement for magnesium and have been found adjacent to one another in their bacterial genomes in all cases studied so far (Promega 1996). • Recognition sites • Restriction enzymes recognise specific sites of different lengths and base composition. The typical restriction enzyme site (type II) is an exact palindrome of 4, 5, 6, 7 or 8 bp with an axis of rotational symmetry (palindromic, recognition sequence, e.g., the EcoR I recognition site is GAATTC). Most enzymes will not cut DNA methylated on one or both strands of their recognition site, although a few require methylation in order to cut DNA. • The number and size of the fragments generated by a restriction enzyme depend on the frequency of occurrence of the restriction enzyme site in the DNA to be cut. Assuming 50% G+C content, a four base recognition site occurs, on the average, once every 44 (256) bp, while a six base recognition site occurs every 46 (4,096) bp and an eight base recognition site occurs every 48 (65,536) bp. DNA is cut into many small pieces with four base restriction enzymes such as Hae III for an application like DNA fingerprinting. DNA is cut into larger fragments with eight base cutters for genomic cloning or mapping. Six base cutters are used for cloning into specific regions of plasmids in which a series of unique sites known as polylinkers (multiple cloning site) have been engineered (Promega 1996). • Type II enzymes have the following important characteristics: • Most restriction enzymes recognise a single restriction site. The restriction site is recognised without regard of the source of the DNA. • Because most restriction enzymes recognise a unique restriction site sequence, the number of cuts in the DNA from a particular organism is determined by the number of restriction sites present (Hartl and Jones 2000). Figure 1. Recognition site of the restriction endonuclease BamH I and resulting restriction fragments after cleavage (Hartl and Jones 2000) page:

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