Cutting and joining DNA molecules:

To construct the plasmid with desire DNA fragment, both the plasmid and the desired DNA fragment has to be digested with the same restriction enzyme. Restriction enzymes are nucleases that cut double stranded DNA at specific nucleotide sequence known as restriction site. Theses enzymes were discovered in early 1960s by Werner Arber as DNA cutting enzymes. These enzymes are isolated by several types of bacterial species. It is interesting to note that a restriction enzyme found in a bacterial species does not cleave its own DNA, as recognition sites of the restriction enzymes are modified in the species by an enzyme called methylases. These restriction endonucleases in bacterial system are thought to have evolved as defense system to fight against foreign DNA that invading the bacterial cell such as viruses. To join the digested plasmid and insert, another enzyme called DNA ligase is require. Hence to generate recombinant DNA molecule two major categories of enzymes are required: restriction endonuclease and DNA ligase. Restriction enzyme cleaves phosphodiester bonds at the specific restriction sites and DNA ligases ligate double stranded DNA molecule by formation of phosphodiester bond between the two distinctly originated DNA molecules, thereby forming the recombinant DNA molecule which is transformed to bacterial cells. Different types of restriction endonucleases with their properties are summarized in Table 2.

Table 2: Classification of restriction endonucleases

Class	Abundance	Recognition site	composition	Use in recombinant DNA technology
Type I	Less common	Cut both strands at a nonspecific location > 1000 bp away from recognition site	Individual recognition, endonuclease, and methylase activity.	Not useful
Type II	More common	Cuts double strand at a specific, usually palindromic, recognition site 4-8 bp	Endonuclease and methylase are separate, single- subunit enzymes.	Very useful
Type III	Rare	Cleaves single strand, 24- 26 bp downstream of the 3' recognition site.	Endonuclease and methylase are separate.	Not useful