Restriction MAP Calculator

Type II restriction endonucleases bind short, usually palindromic DNA sequences and cleave both strands at a fixed offset within or beside that site. The recognition sites in this tool span the common lengths: six-base cutters such as EcoRI (GAATTC), BamHI (GGATCC), HindIII (AAGCTT), XhoI (CTCGAG), SalI (GTCGAC), NcoI (CCATGG), PstI (CTGCAG), SmaI (CCCGGG) and KpnI (GGTACC), plus the eight-base rare cutter NotI (GCGGCCGC). In random DNA of 50 percent GC content, an n-base site is expected once every 4 to the power n bases, so a four-cutter appears about every 256 bp, a six-cutter about every 4,096 bp, and an eight-cutter about every 65,536 bp. NotI is rarer still in practice because its GC-only site is depleted in AT-rich genomes, which is why it is favoured for pulsed-field mapping of megabase fragments. The cut offset determines the end chemistry. EcoRI cuts after the first base to leave a four-base 5-prime overhang AATT; BamHI, HindIII, XhoI, SalI and NcoI likewise leave 5-prime overhangs; PstI and KpnI cut near the 3-prime end of their sites to leave 3-prime overhangs; SmaI cuts dead centre and leaves blunt ends. Fragment arithmetic depends on topology: a linear molecule with n cuts yields n+1 fragments, a circular plasmid yields exactly n, and single-cut plasmids linearise without loss. Two practical caveats bound the prediction. Dam and Dcm methylation in standard E. coli strains blocks or alters several enzymes, and star activity, driven by excess enzyme, glycerol above about 5 percent, or low ionic strength, produces cleavage at degenerate sites the map does not show.

Restriction was first a puzzle in phage genetics: certain bacterial strains destroyed incoming phage DNA while sparing their own. Werner Arber at Geneva proposed in the early 1960s that a paired restriction and modification system, an endonuclease plus a protective methylase, explained the effect, and Matthew Meselson and Robert Yuan purified the first such enzyme from Escherichia coli K in 1968. That enzyme, later classed as Type I, cut at unpredictable distances from its recognition site and was useless for mapping. The breakthrough came in 1970 when Hamilton Smith and Kent Wilcox at Johns Hopkins isolated HindII from Haemophilus influenzae and showed it cleaved at a defined position within a specific sequence. Daniel Nathans, working in the same department, applied these enzymes to simian virus 40 and published the first restriction map of a genome in the early 1970s, establishing the technique this calculator reproduces. Smith and Nathans also proposed the naming convention still in use, with a genus initial, two species letters, a strain designator and a Roman numeral for discovery order. Janet Mertz and Ronald Davis showed in 1972 that EcoRI fragments carry complementary cohesive ends and can be rejoined, and Stanley Cohen and Herbert Boyer exploited that in 1973 to build the first recombinant plasmid. Arber, Nathans and Smith shared the 1978 Nobel Prize in Physiology or Medicine. Richard Roberts began compiling REBASE in 1980, and it remains the authoritative catalogue of recognition sequences and cut positions.