Table 1.
Current DNA assembly methods for the synthesis of large DNA molecules. The table has been reproduced from Nature reviews 14: 781–793, with permission from Nature Publishing Group.
| Method | Mechanism | Overhang (bp) | Scar (bp) | Comments | Examples of applications |
|---|---|---|---|---|---|
| BioBricks | Type IIP restriction endonuclease | 8 | 8 | Sequentially assembles small numbers of sequences | Construction of a functional gene expressing enhanced cyan fluorescent protein |
| BglBricks | Type IIP restriction endonuclease | 6 | 6 | Uses a highly efficient and commonly used restriction endonuclease, the recognition sequences of which are not blocked by the most common DNA methylases | Construction of constitutively active gene-expression devices and chimeric, multidomain protein fusions |
| Pairwise selection | Type IIS restriction endonuclease | 65 | 4 | Requires attachment tags at each end of fragments to act as promoters for antibiotic resistance markers; rapid, as a liquid culture system is used | Assembly of a 91 kb fragment from 1-2 kb fragments |
| GoldenGate | Type IIS restriction endonuclease | 4 | 0 | Allows large-scale assembly; ligations are done in parallel one-step assembly of 2-3 fragment | One-step assembly of 2-3 fragments |
| Overlapping PCR | Overlap | 0 | 0 | Uses overlapping primers for the PCR amplification of 1–3 kb-long fragments | Usually used for 1–3 kb-long fragments, for example, for gene cassette construction |
| CPEC | Overlap | 20–75 | 0 | Uses a single polymerase for the assembly of multiple inserts into any vector in a one-step reaction in vitro | One-step assembly of four 0.17–3.2 kb-long PCR fragments |
| Gateway | Overlap | 20 | 0 | Uses a specific recombinase for small-scale assembly | One-step assembly of three 0.8–2.3 kb-long fragments |
| USER | Overlap | Up to 708 | 0 | Replaces a thymidine with a uracil in the PCR primers, which leaves 3′ overhangs for cloning after cleaving by a uracil exonuclease | One-step assembly of three 0.6–1.5 kb-long fragments |
| InFusion | Overlap | 15 | 0 | Uses an enzyme mix for parallel assembly through a “chew-back-and-anneal” method | One-step assembly of three 0.2–3.8 kb-long fragments |
| SLIC | Overlap | >30 | 0 | (i) Uses a T4 DNA polymerase through a chew-back method in the absence of dNTPs (ii) Uses Recombinase A∗ to stabilize the annealed fragments and avoid in vitro ligation (iii) Allows the parallel assembly of several hundred base-long fragments |
Generation of a ten-way assembly of 300–400 bp-long PCR fragments |
| Gibson | Overlap | 40–400 | 0 | Uses enzymatic “cocktails” to chew back and anneal for the parallel assembly of several kilobase-long fragments | Assembly of the 1.08 Mb Mycoplasma mycoides JCVI-syn1.0 genome |