Restriction Enzyme Finder & Virtual Digest

About the Restriction Enzyme Finder & Virtual Digest

Paste a DNA sequence (plain, FASTA or a GenBank record) and the finder looks on both strands for the recognition sites of 387 restriction enzymes, those REBASE lists as sold by New England Biolabs or Thermo Fisher Scientific, including degenerate sites such as GTMKAC, Type IIS enzymes that cut outside their site such as BsaI, and enzymes that cut on both sides. Linear sequences and circular plasmids are both handled, sites across the origin included.

The list shows each enzyme’s site and cut, how many times it cuts and where, the ends it leaves, and whether Dam, Dcm or CpG methylation overlaps its sites; show the single or double cutters to plan a cloning, or the enzymes that do not cut. Pick enzymes for a virtual digest and you get the fragments with their exact ends (the real 4-base overhangs of a Type IIS enzyme in your sequence), a simulated gel next to a ladder, with a lane for each enzyme alone and one for all together, and a map of the cuts. Everything runs in your browser: the sequence is not uploaded. With a Pro pass, copy the lists or download them as CSV and the fragments as FASTA, or unlock this one result. Without a pass the free preview shows every count (enzymes that cut, cut once or twice, fragments), the first rows of each list (up to 10), and the gel and the map under a MySmartCoPilot mark.

Common uses

• Finding unique sites to open a plasmid for cloning
• Choosing a diagnostic digest that tells two constructs apart on a gel
• Checking the overhangs of a Golden Gate design with BsaI, BsmBI or SapI
• Making sure an insert has no internal site for the enzymes you clone with
• Teaching restriction mapping and gel electrophoresis

How the sites are found

Each recognition site is written in IUPAC codes (R = A or G, Y = C or T, N = any base …) and searched on the top strand and, unless it is its own reverse complement, as its reverse complement for the bottom strand. A base of your sequence matches a code only when every base it may be is allowed there, so an N in your sequence never makes a site appear. On a circle the search wraps through the origin. Enzymes with the same site and the same cut (isoschizomers) are listed once, under the name REBASE lists for the most suppliers, with the others named under it.

Cut positions and ends

A cut position is counted on the top strand: “after 396” means between bases 396 and 397. A site shown as G^AATTC is cut after the G on both strands’ readings; a site shown as GGTCTC(1/5) is cut 1 base after the site on the top strand and 5 on the bottom one, which leaves a 5′ overhang of 4 bases taken from your own sequence; (7/12)GAANNNNNNNTTGG(11/6) means cuts on both sides of the site. A site too close to the end of a linear sequence to be cut is counted but gives no cut.

Methylation

Many E. coli strains used for cloning methylate the A of GATC (Dam) and the inner C of CCWGG (Dcm), and mammalian DNA carries 5-methylcytosine at CpG. Whether a methylated base inside a site blocks an enzyme, slows it or makes no difference depends on the enzyme. The list therefore says where Dam, Dcm or CpG methylation overlaps each enzyme’s sites, and you check the supplier’s methylation-sensitivity chart before digesting plasmid from a dam⁺ or dcm⁺ strain or methylated genomic DNA.

The simulated gel

Bands are spaced by the logarithm of their size between the ladder’s largest and smallest bands, a common first approximation for linear DNA on agarose. It shows the pattern to expect, not exact positions: real migration depends on the agarose percentage, the buffer and the voltage, very large and very small fragments flatten out, and supercoiled, nicked and linear forms of an uncut plasmid run differently. Bands much smaller than the largest fragment are drawn fainter, because an equal number of molecules of a short fragment carries less DNA.

Sources

• REBASE, the restriction enzyme database (Roberts R.J., Vincze T., Posfai J. and Macelis D., Nucleic Acids Research 51:D629): the recognition sites, cut positions and suppliers, as compiled in Biopython’s restriction dictionary.
• IUPAC-IUB nucleotide codes, as in the reverse complement generator.

Limitations

• Only the 387 enzymes REBASE lists as sold by New England Biolabs or Thermo Fisher Scientific are searched; enzymes that cut only methylated DNA (such as DpnI) are left out.
• Methylation is reported as an overlap with the site, not as each enzyme’s sensitivity: check the supplier’s chart.
• Star activity, partial digests, buffer compatibility and the distance an enzyme needs from the end of a fragment are not modelled.
• The gel is a sketch on a logarithmic scale, not a prediction of a real gel.
• Sequences up to 1,000,000 bases; for very frequent cutters the list shows the first cut positions and the CSV all of them.

Privacy

On this device: nothing is sent

Everything happens in your browser. What you enter or open here is not uploaded or stored by MySmartCoPilot.