Seamless assembly
A single 50 °C reaction containing a 5' exonuclease, a high-fidelity polymerase and a thermostable ligase joins pieces that share designed terminal overlaps, usually 20–25 bp added through PCR primers, with no restriction site involved and no scar left at the junction.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Gibson
A single 50 °C reaction containing a 5' exonuclease, a high-fidelity polymerase and a thermostable ligase joins pieces that share designed terminal overlaps, usually 20–25 bp added through PCR primers, with no restriction site involved and no scar left at the junction.
How it differs from the standard method
- Overlaps need to stay annealed at 50 °C: 20–25 bp at moderate GC is the usual target, and 15 bp is marginal.
- Two or three pieces assemble in 15 min; four or more need an hour so that every overlap is exposed.
- Only the ends matter, so internal restriction sites, repeats within a fragment and awkward sequence are irrelevant.
- Every piece is a PCR product: use a proofreading polymerase, keep cycles down, DpnI the template and sequence the junctions.
- Overlaps that resemble one another are the main design failure, giving fragments in the wrong order.
1The junction is sequence you choose
Ligation-based cloning takes the junctions the restriction enzymes give it, and leaves their recognition sites behind in the product. Seamless assembly inverts that.
2Overlap assembly: chew back, anneal, fill in, seal
In Gibson-type assembly each piece is made with 15–40 bp of its neighbour's sequence on the end, usually added through the PCR primers. One enzyme mix does three things at 50 °C: a 5' exonuclease chews back the 5' strands to expose complementary 3' single strands, those single strands anneal to each other, then a polymerase fills the remaining gaps and a ligase seals the nicks.
3Golden Gate: cutting outside the recognition site
Type IIS enzymes such as BsaI and BsmBI bind one sequence and cut a fixed distance away from it, so the recognition site stays on the offcut and disappears from the product. Two things follow.
Gibson / In-Fusion
Junctions are designed overlaps added by PCR. Most flexible, no sites needed, but every piece is a PCR product with the error rate that implies.
Golden Gate
Junctions are defined four-base overhangs. One pot, ordered, reusable parts, and no new PCR errors, if the parts are domesticated.
Gateway
Junctions are fixed att sites. Fastest and most reproducible, but the sites stay in the construct and the vectors are not yours to design.
4Recombination: fast, fixed, and not quite scarless
Site-specific recombinases move a fragment between vectors without cutting or ligating anything you control. Lambda-derived clonase mixes recombine attB with attP (the BP reaction, giving an entry clone) and attL with attR (the LR reaction, giving an expression clone), and no other combination does anything at all.
5Where assemblies go wrong
- Mixing by mass instead of by moles: equal masses of unequal pieces are unequal numbers of molecules, and the longest piece quietly becomes limiting. Convert each piece to pmol (ng × 1000 / (660 × bp)) and mix equimolar.
- Overlaps or overhangs that resemble each other: the method matches pieces by sequence, so two similar junctions let fragments swap places or drop out. Keep every junction distinct, and watch for repeats elsewhere in the construct.
- Mutations introduced by the PCR that made the fragments: every base of an overlap assembly passes through a polymerase, so a high-fidelity enzyme and a modest cycle number matter, and the junctions must be sequenced.
- The plasmid the fragments were amplified from: it is circular and supercoiled, so it transforms far better than any assembly product. Digest it with DpnI, which cuts methylated template and ignores the PCR product.
- Too few junctions screened: a missing piece, a reversed piece and a tandem copy all give colonies, and all of them look fine on a single colony PCR.
- Few colonies, mostly incomplete
- Junctions are forming only sometimes: longer overlaps, a full hour at 50 °C, or digest/ligate cycling.
- Many colonies, all empty backbone
- Background, not assembly. Check the backbone-only control, the DpnI step and, for Golden Gate, that an internal site is not cutting a part.
- Right size, wrong sequence at a junction
- Polymerase errors in the amplified pieces, or a mispaired overlap. Sequence every junction and compare the fragments' own sequence.
- Nothing at all, competence control fine
- A piece is missing from the tube, the att sites are not a pair, or the enzyme mix has been through too many freeze–thaw cycles.
Key terms
Practise these as flashcards →The 15–40 bp of shared sequence at the ends of two neighbouring pieces, which defines their junction in overlap assembly.
A restriction enzyme that cuts a fixed distance outside its recognition sequence, so the site is removed from the product.
Silently removing internal copies of the assembly enzyme's recognition site from a part, so only the intended ends are cut.
The fixed recombination sites of phage lambda and the enzyme mix that recombines them: attB × attP in a BP reaction, attL × attR in an LR reaction.
A toxic gene between a vector's att sites; cells that take up unreacted vector are killed, so most colonies carry a real product.
Sequence left at a junction by the cloning method itself rather than chosen by the designer.
Equal numbers of molecules of every piece, which is not the same as equal masses.
An enzyme that cuts Dam-methylated DNA, used to destroy the plasmid template a fragment was amplified from.
When to use it
- Joining three or more pieces in one reaction, where sequential ligations would take weeks.
- There is no usable pair of restriction sites, or every candidate site also occurs inside the insert.
- The junction itself matters: a protein fusion with no linker, or a promoter placed exactly against a start codon.
- Building a library of variants at one junction, or a family of constructs that reuse the same parts.
- Moving one insert into many different vectors, which is what recombination systems are built for.
Compared with alternatives
Cheaper and perfectly good for a single insert into a vector you already cut; prefer it when the sites exist and a scar does not matter.
Still how a backbone is usually linearised, and how you verify an assembly afterwards with a diagnostic digest.
Prefer it for changing a few bases in an existing plasmid; assembly is for building a new arrangement of pieces.
Supplies the overlaps and the fragments, and is also the source of the junction mutations you have to sequence for.
What turns the assembled circles into colonies; its efficiency and the plate's selection decide what you see.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A single 50 °C reaction containing a 5' exonuclease, a high-fidelity polymerase and a thermostable ligase joins pieces that share designed terminal overlaps, usually 20–25 bp added through PCR primers, with no restriction site involved and no scar left at the junction.
- Overlaps need to stay annealed at 50 °C: 20–25 bp at moderate GC is the usual target, and 15 bp is marginal.
- Two or three pieces assemble in 15 min; four or more need an hour so that every overlap is exposed.
- Only the ends matter, so internal restriction sites, repeats within a fragment and awkward sequence are irrelevant.
- Every piece is a PCR product: use a proofreading polymerase, keep cycles down, DpnI the template and sequence the junctions.
- Overlaps that resemble one another are the main design failure, giving fragments in the wrong order.