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Assembling Gene Fragments using Isothermal Assembly

gBlocks® Gene Fragments

gBlocks® Gene Fragments are high fidelity, double-stranded DNA (dsDNA) up to 500 bp that can be custom designed for virtually any cloning application, and are uniquely suited for assembling and modifying larger constructs using the Gibson Assembly™ method. gBlocks Gene Fragments provide flexibility for synthetic biology applications that is not currently available in any other product.

The Gibson isothermal assembly method is based on the technique described by Gibson et al. in Nature [1]. The method relies on the use of an enzyme mixture consisting of the mesophilic T5 exonuclease, a thermophilic ligase, and a high fidelity polymerase. Both the vector and the 5’ ends of
the gBlocks fragments to be assembled are designed with 30 bp overlaps. Prior to assembly, the vector must be linearized by a separate restriction digestion or PCR amplification.

For assembly, all of the components are combined and allowed to react at 50°C, where the exonuclease begins to digest dsDNA from the 5’ ends. The exonuclease is rapidly inactivated by the high temperature, leaving complementary 3’ single-stranded DNA (ssDNA) ends that anneal. The polymerase then fills in any remaining ssDNA gaps and the ligase covalently joins the fragments together (See Figure 1 in the article, Isothermal Assembly: Quick, Easy Gene Construction in the January 2012 issue of the DECODED newsletter).

Isothermal assembly of gBlocks Gene Fragments makes it easy to create or modify genes without the need for restriction sites in your insert or for conducting time-consuming, sequential, cloning reactions. For more information on gBlocks Gene Fragments, see New! Easy Gene Assembly in this issue.

References

  1. Gibson DG, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.

Now read about how gBlocks Gene Fragments are used in: Building Biological Factories for Renewable and Sustainable Products,
or browse the current issue of the DECODED newsletter.