Restriction enzymes and cut sites, explained
Restriction enzymes are molecular scissors that cut DNA at specific sequences. Knowing where they cut — and what kind of ends they leave — is the foundation of traditional cloning.
What an enzyme recognises
A restriction enzyme binds a short, specific stretch of double-stranded DNA called its recognition site — typically 4 to 8 base pairs — and cuts both strands at a fixed position relative to it. Most common sites are palindromes: the sequence reads the same 5′→3′ on both strands, so the enzyme cuts wherever that sequence appears, on either strand.
Sticky ends vs blunt ends
The position of the cut determines the ends you get. A staggered cut — offset between the two strands — leaves short single-stranded overhangs, called sticky ends. Overhangs are handy in cloning because two fragments cut with the same enzyme have complementary overhangs that base-pair and ligate together easily. A straight cut through both strands at the same place leaves blunt ends (for example SmaI, CCC▾GGG), which ligate in any orientation but less efficiently.
Worked example: EcoRI
EcoRI recognises GAATTC and cuts after the first base — between the G and the first A — on each strand:
5′-G▾A A T T C-3′
3′-C T T A A▾G-5′
Because the two strands are cut at offset positions, EcoRI leaves a 5′ overhang of AATT on each fragment — sticky ends. Any two pieces of DNA cut with EcoRI share that AATT overhang, so they can be joined to one another. (This is exactly the recognition sequence and cut position the calculator uses.)
Why it matters for cloning
When you plan a digest, you usually want enzymes whose sites flank your insert but do not also appear inside your gene of interest — an internal site would chop the very fragment you are trying to move. Scanning your sequence for each candidate enzyme’s sites first tells you which enzymes are safe to use and where they cut.
The calculator covers a curated, commonly used subset of enzymes, not the full REBASE database — enough for everyday cloning checks, but always confirm against your enzyme supplier for anything unusual.