Gibson assembly
Gibson assembly is a molecular cloning method which allows for the joining of multiple DNA fragments in a single, isothermal reaction. It was invented in 2009 by Daniel Gibson while he was at the J. Craig Venter Institute (JCVI).[1]
Process
The entire Gibson assembly reaction requires few components with minor manipulations.[1][2]
The method can simultaneously combine more than ten DNA fragments based on sequence identity. It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. These DNA fragments are mixed with a cocktail of three enzymes, along with other buffer components.
The three required enzyme activities are: exonuclease, DNA polymerase, and DNA ligase.
- The exonuclease chews back DNA from the 5' end. The resulting single-stranded regions on adjacent DNA fragments can anneal.
- The DNA polymerase incorporates nucleotides to fill in any gaps.
- The DNA ligase covalently joins the DNA of adjacent segments, thereby removing any nicks in the DNA.
The entire mixture is incubated at 50 °C for up to one hour. The resulting product is different DNA fragments joined into one.
Advantages
This DNA assembly method has many advantages compared to conventional restriction enzyme/ligation cloning of recombinant DNA.
- No restriction digest of the DNA fragments after PCR is necessary. However, the backbone vector can be digested, or synthesized by PCR.
- It is cheaper and faster than conventional cloning schemes, as it requires fewer steps and fewer reagents.
- No restriction site scar remains between two DNA fragments, but the region between the double strands and hanging ends is slightly susceptible to mutation when DNA polymerase closes the gaps.
- More than 10 DNA fragments can be combined simultaneously in a single-tube reaction.
References
- 1 2 Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods. 6 (5): 343–345. doi:10.1038/nmeth.1318. PMID 19363495.
- ↑ Gibson DG. (2011). "Enzymatic assembly of overlapping DNA fragments". Methods in Enzymology. 498: 349–361. doi:10.1016/B978-0-12-385120-8.00015-2. PMID 21601685.
Further information
- A Guide to Gibson Assembly from the University of Cambridge, UK
- Gibson Assembly - A tutorial from supplier New England Biolabs
- Perkel, Jeffrey M. (January 2014). "Seamlessly rewriting the lab cloning manual". Tech News. BioTechniques. 56 (1): 12–14.
Gibson says he no longer even bothers with standard restriction enzyme-based cloning in his lab.
