Restriction Mapping
Restriction enzymes are bacterial proteins that cut DNA at short, specific recognition sequences. A restriction map lists where in your sequence each enzyme would cut. It is the starting point for cloning, for checking a plasmid by digestion and for planning a diagnostic digest.
Restriction enzyme recognition sites and cut positions
Most enzymes recognise a palindrome: the site reads the same 5'→3' on both strands, like GAATTC, because the enzyme is a symmetric dimer. They cut each strand at a fixed spot in the site. The marker ^ shows the cut: EcoRI is G^AATTC, BamHI G^GATCC, HindIII A^AGCTT.
Sticky and blunt ends
If the two cuts are staggered, each fragment gets a short single-stranded overhang, a sticky end (EcoRI leaves a 5' AATT overhang; PstI, CTGCA^G, leaves a 3' overhang). Sticky ends with matching overhangs ligate easily and in a defined orientation. Enzymes that cut straight through (SmaI, CCC^GGG, and EcoRV, GAT^ATC) leave blunt ends, which ligate less efficiently and in either orientation. Different enzymes can leave compatible ends: BamHI and BglII both leave a GATC overhang and can be joined, although the joint is no longer cut by either.
Common restriction enzymes
| Enzyme | Recognition site (^ = cut) | Ends |
|---|---|---|
| EcoRI | G^AATTC | 5' overhang AATT |
| BamHI | G^GATCC | 5' overhang GATC |
| HindIII | A^AGCTT | 5' overhang AGCT |
| XhoI | C^TCGAG | 5' overhang TCGA |
| NcoI | C^CATGG | 5' overhang CATG |
| XbaI | T^CTAGA | 5' overhang CTAG |
| PstI | CTGCA^G | 3' overhang TGCA |
| KpnI | GGTAC^C | 3' overhang GTAC |
| SmaI | CCC^GGG | blunt |
| EcoRV | GAT^ATC | blunt |
| NotI | GC^GGCCGC | 5' overhang GGCC |
Counting fragments: linear and circular DNA
A linear piece of DNA cut at n sites gives n + 1 fragments. A circular plasmid cut at n sites gives n fragments, because the first cut only opens the circle. So one EcoRI site turns a 3,000 bp plasmid into a single 3,000 bp linear piece, and two sites 1,000 bp apart give 1,000 and 2,000 bp.
Planning a digest
For cloning a fragment into a vector you need sites that (1) are in your insert's flanking region, (2) are in the vector's multiple cloning site and (3) do not cut inside the part you want to keep. A unique cutter, an enzyme with exactly one site, is the natural choice to open a plasmid. Using two different enzymes gives a directional insert.
How Aaron's Archive does it
| Item | What the tool does |
|---|---|
| Enzymes | 36 commonly used enzymes with 4- to 8-base palindromic sites, including EcoRI, BamHI, HindIII, XhoI, NotI, SmaI, PstI and EcoRV. |
| Output | A visual map with one row per cutting enzyme, and a table with the recognition sequence, the number of cuts and the positions. |
| Fragment sizes | Subtract neighbouring cut positions to get fragment lengths. |
Pitfalls
The sequence is treated as linear: a site that spans the join of a circular plasmid is not detected. The tool looks for the sequence only; it does not know about methylation (some enzymes, for example ClaI and XbaI in some contexts, are blocked by Dam methylation of plasmids grown in E. coli), buffers or star activity (cutting at similar-but-wrong sites in poor conditions). Check the supplier's catalogue before you plan an experiment around a map.
Common questions about restriction mapping
What is a recognition site?
The short DNA sequence, usually 4 to 8 bases, that a restriction enzyme binds and cuts, such as GAATTC for EcoRI.
What is the difference between sticky and blunt ends?
Sticky ends have a short single-stranded overhang that pairs with a matching overhang, so they join easily and in a set orientation. Blunt ends have no overhang and join less efficiently, in either orientation.
How do I find an enzyme that cuts only once?
Run a restriction map and look for enzymes with exactly one site. Such a unique cutter is the usual way to open a plasmid.
Where can I map restriction sites?
Use the restriction site finder: it searches 36 common enzymes and lists the site and cut positions.