Role of Recombinant DNA Technology to Improve Life
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 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
