Restriction Enzymes — Recognition Sites & Cut Positions
A restriction enzyme recognises a specific short DNA sequence — its recognition site — and cuts the double helix there. This table lists 49 commonly used enzymes with their recognition sequences (5′→3′), the cut position on the top strand (marked ▾), and whether they leave blunt or sticky ends.
| Enzyme | Recognition site (5′→3′) | Length | Ends |
|---|---|---|---|
| AflII | C▾TTAAG | 6 bp | 5′ overhang (4) |
| AgeI | A▾CCGGT | 6 bp | 5′ overhang (4) |
| AluI | AG▾CT | 4 bp | Blunt |
| ApaI | GGGCC▾C | 6 bp | 3′ overhang (4) |
| ApoI | R▾AATTY | 6 bp | 5′ overhang (4) |
| AscI | GG▾CGCGCC | 8 bp | 5′ overhang (4) |
| AvrII | C▾CTAGG | 6 bp | 5′ overhang (4) |
| BamHI | G▾GATCC | 6 bp | 5′ overhang (4) |
| BglII | A▾GATCT | 6 bp | 5′ overhang (4) |
| BspEI | T▾CCGGA | 6 bp | 5′ overhang (4) |
| BstBI | TT▾CGAA | 6 bp | 5′ overhang (2) |
| BstYI | R▾GATCY | 6 bp | 5′ overhang (4) |
| ClaI | AT▾CGAT | 6 bp | 5′ overhang (2) |
| DdeI | C▾TNAG | 5 bp | 5′ overhang (3) |
| DraI | TTT▾AAA | 6 bp | Blunt |
| EagI | C▾GGCCG | 6 bp | 5′ overhang (4) |
| EcoRI | G▾AATTC | 6 bp | 5′ overhang (4) |
| EcoRV | GAT▾ATC | 6 bp | Blunt |
| FseI | GGCCGG▾CC | 8 bp | 3′ overhang (4) |
| HaeIII | GG▾CC | 4 bp | Blunt |
| HindIII | A▾AGCTT | 6 bp | 5′ overhang (4) |
| HinfI | G▾ANTC | 5 bp | 5′ overhang (3) |
| HpaI | GTT▾AAC | 6 bp | Blunt |
| KpnI | GGTAC▾C | 6 bp | 3′ overhang (4) |
| MboI | ▾GATC | 4 bp | 5′ overhang (4) |
| MluI | A▾CGCGT | 6 bp | 5′ overhang (4) |
| MspI | C▾CGG | 4 bp | 5′ overhang (2) |
| NcoI | C▾CATGG | 6 bp | 5′ overhang (4) |
| NdeI | CA▾TATG | 6 bp | 5′ overhang (2) |
| NheI | G▾CTAGC | 6 bp | 5′ overhang (4) |
| NotI | GC▾GGCCGC | 8 bp | 5′ overhang (4) |
| NsiI | ATGCA▾T | 6 bp | 3′ overhang (4) |
| PacI | TTAAT▾TAA | 8 bp | 3′ overhang (2) |
| PmeI | GTTT▾AAAC | 8 bp | Blunt |
| PstI | CTGCA▾G | 6 bp | 3′ overhang (4) |
| PvuII | CAG▾CTG | 6 bp | Blunt |
| SacI | GAGCT▾C | 6 bp | 3′ overhang (4) |
| SalI | G▾TCGAC | 6 bp | 5′ overhang (4) |
| SbfI | CCTGCA▾GG | 8 bp | 3′ overhang (4) |
| ScaI | AGT▾ACT | 6 bp | Blunt |
| ScrFI | CC▾NGG | 5 bp | 5′ overhang (1) |
| SmaI | CCC▾GGG | 6 bp | Blunt |
| SpeI | A▾CTAGT | 6 bp | 5′ overhang (4) |
| SphI | GCATG▾C | 6 bp | 3′ overhang (4) |
| StuI | AGG▾CCT | 6 bp | Blunt |
| SwaI | ATTT▾AAAT | 8 bp | Blunt |
| TaqI | T▾CGA | 4 bp | 5′ overhang (2) |
| XbaI | T▾CTAGA | 6 bp | 5′ overhang (4) |
| XhoI | C▾TCGAG | 6 bp | 5′ overhang (4) |
▾ marks the top-strand cut. Overhang length in parentheses. Enzymes shown are palindromic Type IIP cutters.
Blunt vs sticky ends
When an enzyme cuts both strands at the same position it produces blunt ends (e.g. SmaI, EcoRV). When it cuts the two strands at offset positions it leaves short single-stranded overhangs — sticky or cohesive ends. A 5′ overhang (e.g. EcoRI) leaves the single strand on the 5′ side; a 3′ overhang (e.g. PstI, KpnI) on the 3′ side. Sticky ends base-pair with complementary overhangs, which makes directional cloning and ligation more efficient.
4-, 6- and 8-cutters
The length of the recognition site determines how often an enzyme cuts. A 4-cutter (4 bp site, e.g. AluI, HaeIII) cuts on average every ~256 bp; a 6-cutter (e.g. EcoRI, BamHI) about every ~4 kb; an 8-cutter (e.g. NotI, AscI) very rarely, which is useful when you need a single cut in a large construct.
Frequently asked questions
- What is a restriction enzyme?
- A restriction enzyme (restriction endonuclease) is a protein that recognises a specific short DNA sequence — its recognition site — and cuts the double helix at or near that site. Bacteria use them as a defence against viral DNA; molecular biologists use them to cut and join DNA fragments.
- What is the difference between blunt and sticky ends?
- If an enzyme cuts both strands at the same position it leaves blunt ends. If it cuts the two strands at offset positions it leaves short single-stranded overhangs — sticky (cohesive) ends — that base-pair with complementary overhangs, making ligation more efficient.
- What is a 6-cutter?
- It refers to the length of the recognition site. A 6-cutter recognises a 6 bp sequence and so cuts on average every ~4,096 bp; a 4-cutter (4 bp site) cuts much more often (~every 256 bp), and an 8-cutter cuts rarely. Longer sites mean fewer, more specific cuts.
- What does the ▾ symbol mean in the recognition site?
- It marks where the enzyme cuts the top (5′→3′) strand within the recognition sequence. For example EcoRI G▾AATTC cuts between G and A on the top strand, leaving a 4-base 5′ overhang.
- Are these the only restriction enzymes?
- No — there are thousands. This is a curated set of enzymes commonly used in cloning. For a comprehensive catalogue see a supplier database such as REBASE.