BioDeviceHub

Restriction Site Finder

Find where named restriction enzymes cut a DNA sequence, linear or circular, with each enzyme's real recognition sequence, cut positions, overhang type and unique cutters.

Formula

sites = every match of the enzyme’s recognition sequence on either strand\text{sites = every match of the enzyme's recognition sequence on either strand}
cut position = bases of the top strand to the left of the cut\text{cut position = bases of the top strand to the left of the cut}
∧\mathord{\wedge}
marks where the enzyme cuts the top strand within its recognition sequence; Type IIS enzymes are written as site(n/m), the two cuts lying n and m bases beyond it
overhang\text{overhang}
bases left single-stranded at each cut end: 5′, 3′ or none (blunt)

How it works

Each restriction enzyme recognises a specific short sequence and cuts at a fixed position in or near it. The tool searches both strands for each enzyme's recognition sequence, using the degenerate letters some sites contain (HincII recognises GTYRAC), and converts each site to a cut position on the top strand.

The result shows which enzymes are single cutters, which do not cut, and what kind of end each leaves. EcoRI and XmaI leave 5′ overhangs, PstI and KpnI leave 3′ overhangs, and SmaI leaves a blunt end. Enzymes are not interchangeable even when they share a recognition sequence: SmaI and XmaI both recognise CCCGGG, but cut it differently and cannot be swapped where sticky ends are needed.

Worked example

A 57 bp cloning-site region, tested with EcoRI, BamHI, HindIII, PstI, KpnI and NotI, as a linear sequence.

  1. Each of the first five finds exactly one site.
  2. EcoRI cuts after base 52, BamHI after 31, PstI after 17, KpnI after 44 and HindIII after base 1.
  3. NotI (GCGGCCGC) is not present.

Five of six enzymes cut, each once, so all five are single cutters. NotI does not cut.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The sequence is searched as written, with no methylation.
  • Only the enzymes in the list are available, a selection of commonly used ones.
  • A site in a linear sequence counts only if there are flanking bases for both strand cuts.

Common mistakes

  • Assuming a methylation-sensitive enzyme will cut a site in DNA from a dam+ or dcm+ strain. Some enzymes, such as ClaI and XbaI, are blocked when their site overlaps a Dam site.
  • Using a blunt and a sticky cutter together and expecting the ends to ligate to each other.
  • Choosing an enzyme that also cuts inside the insert. Check every enzyme against the whole sequence.

Related tools

Restriction Digest PredictorFragment sizes for a single or multiple enzyme digestOpen calculatorRestriction Digest Reaction SetupVolumes of DNA, buffer, enzymes and water for a digestOpen calculatorLigation Calculator (Vector:Insert Ratio)Insert mass for a molar ratio, or the ratio from the masses you haveOpen calculatorReverse Complement ToolReverse, complement or reverse-complement a sequenceOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.