Gibson assembly
Encyclopedia
Gibson assembly is a DNA assembly method which allows for the joining of multiple DNA fragments in a single, isothermal reaction. It was invented in 2009 by the Daniel Gibson while he was at the J. Craig Venter Institute
(JCVI).
The method can simultaneously combine numerous (>10) DNA fragments based on sequence homology. 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 enzymes are: T5 exonuclease
, a thermostable DNA polymerase
(such as Phusion polymerase), and Taq DNA ligase
.
The entire mixture is incubated at 50°C for up to one hour. The resulting product can be directly transformed into E. coli.
J. Craig Venter Institute
The J. Craig Venter Institute is a non-profit genomics research institute founded by J. Craig Venter, Ph.D. in October 2006. The Institute was the result of consolidating four organizations: the Center for the Advancement of Genomics, The Institute for Genomic Research, the Institute for...
(JCVI).
Process
The entire Gibson assembly reaction requires a small number of components with very few manipulations.The method can simultaneously combine numerous (>10) DNA fragments based on sequence homology. It requires that the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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 enzymes are: T5 exonuclease
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
, a thermostable DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
(such as Phusion polymerase), and Taq DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
.
- The T5 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 Taq 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 can be directly transformed into E. coli.
Advantages of Gibson assembly
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. The backbone vector can be digested, or synthesized by PCR.
- It is far more simple than conventional cloning schemes, as it requires fewer steps and fewer reagents. The process also takes less time.
- No restriction site scar remains between two DNA fragments.
- Multiple DNA fragments can be combined simultaneously in a single reaction.