Restriction Endonucleases and Their Use

      Restriction endonucleases (REs) are bacterial enzymes that cleave double-stranded DNA.  Type I REs are important in bacterial function but do not cleave DNA at specific sequences. Type II restriction endonucleases require highly specific sites for DNA cleavage . These enzymes allow cloning and purification (see section cloning) as well as sequence determination (RFLP, see section rflp). The 400 or so known REs are typically isolated from a variety of bacterial strains and available commercially[80].

Restriction endonucleases are present in bacteria presumably to destroy DNA from foreign sources (e.g., infecting bacteriophage) by cleaving the foreign DNA at specific restriction sites. The host bacteria DNA is protected from cleavage because specific recognition sites are modified, usually by methylation  at one of the bases in the site, making the site no longer a substrate for RE cleavage. Host bacteria used to propagate cloned DNA in the laboratory are usually mutant in the host restriction genes; thus their intracellular enzyme activities will not destroy the foreign recombinant sequences.

The cleavage site specifities for many REs have been defined. They cut DNA within or near to their particular recognition sequences, which typically are four to six nucleotides in length with a twofold axis of symmetry. For example, the RE EcoRI, used for cloning of the 2ar gene (see section cloning), requires that six base pairs occur in the following specific order:

5' 3'
3' 5'

EcoRI recognizes this sequence and cleaves it in a unique fashion, resulting in two termini with protruding 5' ends:

1.5exand

These ends are complementary ("sticky")  and can be enzymatically reattached to any other EcoRI generated termini using T4 DNA ligase  (see ligation).   Many restriction enzymes, like EcoRI, generate fragments with protruding 5' tails; others (e.g., PstI) generate fragments with 3' protruding, cohesive termini, whereas still others (e.g., BalI) cleave at the axis of symmetry to produce blunt-ended fragments.   Each restriction endonuclease  has a specific sequence and number of nucleotides required to create the recognition site. Some REs do not require a specific nucleotide in every position of the recognition site.

REs are useful because their specifity and the resulting ligatable termini allow dissection, analysis, and restructuring of DNA in a controlled, predictable, site-specific manner. The amount of RE activity is not usually defined using classical enzyme kinetics. Rather, RE activity   is defined in practical terms for use in the laboratory. One unit (U) of activity for an restriction endonuclease is defined as the amount of enzyme that will cut 1 g at all specific sites in a DNA sample (usually bacteriophage  ) in 1 hour at 37psy176 C (or the appropriate temperature maximizing enzyme activity). The actual activity achieved may be different if additional RE digestion sites are present.

Analytical digests were performed in a volume of 20 l (approximately 0.2 to 1 g DNA). For preparative digests  , the amount of DNA and the used digestion volume was increased appropriately. The amount of added enzyme and the incubation length was adjusted to the amount of digestible DNA. Manufacturers recommendations for incubation temperature were followed strictly. Only the shipped tenfold buffers were used in digestion assays. For the specific restriction sites of the used enzymes, see section rflpsites.