Therapeutic products

Vaccines Genetically modified products

Growth hormones Fruits
GM vegetables
Antibodies
GM crops
Vectors
GM microbes
Recombinant protein GM animals
Anticancer drugs

Recombinant DNA technology

Diagnosis Energy applications

Gene therapy Biohydrogen
CRISPR Bioethanol
Monitoring devices Biomethanol
Therapeutic strategies Biobutanol
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.
Overhang Scar
Method Mechanism Comments Examples of applications
(bp) (bp)
Construction of a
Type IIP restriction Sequentially assembles small numbers of functional gene expressing
BioBricks endonuclease 8 8 sequences enhanced cyan fluorescent
protein

Construction of
Uses a highly efficient and commonly used
constitutively active gene-
Type IIP restriction restriction endonuclease, the recognition
BglBricks 6 6 expression devices and
endonuclease sequences of which are not blocked by the most
chimeric, multidomain
common DNA methylases protein fusions
Requires attachment tags at each end of fragments Assembly of a 91
Pairwise Type IIS restriction
65 4 to act as promoters for antibiotic resistance kb fragment from
selection endonuclease
markers; rapid, as a liquid culture system is used 1-2 kb fragments
Type IIS restriction Allows large-scale assembly; ligations are done in One-step assembly of
GoldenGate 4 0
endonuclease parallel one-step assembly of 2-3 fragment 2-3 fragments
Usually used for
Overlapping Uses overlapping primers for the PCR 1–3 kb-long fragments,
Overlap 0 0
PCR amplification of 1–3 kb-long fragments for example, for gene
cassette construction
Uses a single polymerase for the assembly of One-step assembly of
CPEC Overlap 20–75 0 multiple inserts into any vector in a one-step four 0.17–3.2 kb-long
reaction in vitro PCR fragments
Uses a specific recombinase for small-scale One-step assembly of three
Gateway Overlap 20 0
assembly 0.8–2.3 kb-long fragments
Replaces a thymidine with a uracil in the PCR One-step assembly of three
USER Overlap Up to 708 0 primers, which leaves 3 overhangs for cloning
0.6–1.5 kb-long fragments
after cleaving by a uracil exonuclease