Restriction enzymes are enzymes that cut both DNA of one and double chain. Each restriction enzyme has a specific nucleotide sequence, called a restriction site, that recognizes and cuts. Restriction enzymes are used for DNA sequencing, mutational analysis, and cloning and DNA amplification. Scientists use restriction enzymes to insert genes of interest into expression vectors, DNA molecules that can be replicated separately from chromosomal DNA. The vector containing the gene of interest can then be introduced into a bacterial strain for the expression and characterization of proteins.
Identify restriction enzyme sites in your vector by looking at a restriction map. The restriction map will tell you which enzymes will cut your vector, and where.
Choose a restriction enzyme that also has a site present in the insert of the gene, by observing the sequence of the insert. Make sure that the restriction site is in a position in the insertion that is outside the gene of interest, so you do not lose any part of the gene.
Make sure there are no duplicates of the restriction site anywhere in your gene insertion or vector. This will cause multiple cuts in your DNA and give you deceptive data.
Try to choose the restriction enzymes that cut cohesive ends, rather than blunt ends. Sticky ends occur when the enzyme cuts double-stranded DNA in a staggered fashion, leaving a single cantilever strand that facilitates fixation with an insertion cut in the opposite manner. Obtuse ends occur when double-stranded DNA is cut in a soft manner, and these are more difficult to join.
Choose a different restriction enzyme for the two ends of your insert to ensure that it is inserted into the vector in the proper orientation and ensure that the vector does not reattach itself.
Try to choose two restriction enzymes that work well in the same buffer and temperature system. If this is not possible, run each digestion separately.
Some web programs have been developed to identify the restriction sites of a DNA sequence quickly. One of these programs is from New England Biolabs.