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Gibson assembly

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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).

Contents

Process

The entire Gibson assembly reaction requires few components with minor manipulations.

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

    Gibson assembly Wikipedia
    (Text) CC BY-SA


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