Site-directed mutagenesis (SDM) is a commonly used technique for introducing mutations into a gene of interest. Existing techniques for SDM, such as whole plasmid SDM, while effective, are time consuming and prone to off-target mutation incorporation. Further, verification of mutation incorporation can be difficult when the insertion site sequence lacks convenient restriction sites for analysis. This can be a serious impediment to the planning and execution of SDM experiments.
Gibson Assembly™, developed by Daniel Gibson and his colleagues at the J. Craig Venter Institute, is a rapid and reliable method for the assembly of DNA fragments in a single-tube, isothermal reaction without reliance upon the presence of restriction sites within the target sequence. The technique, which involves the design of complimentary flanking primers to align fragments, can be readily adapted for SDM applications. In addition, it is unnecessary to use phosphorylated primers for Gibson Assembly, reducing both cost and time.
In one step, Gibson Assembly can assemble two or more PCR products with overlapping ends together or into a pre-cut vector. An exonuclease creates single-stranded 3´ overhangs that promote annealing of complementary fragments at the overlap region. A polymerase then fills in the gaps, which are sealed by the DNA ligase. By introducing multiple complementary mutations in the primers at the overlap region, the Gibson Assembly Master Mix forms a single, covalently bonded DNA molecule that contains the desired mutations and can be directly transformed into competent cells and screened or sequenced.
Here we describe the use of New England Biolabs’ Gibson Assembly Master Mix in two different mutagenesis experiments: first, multiple mutations of the lacZ gene (Figure 1) and second, the mutation of 5 of the 6 nucleotides at position 174–179 of eGFP from CTGACC to TTCTAT in order to change the amino acid sequence from LeuThr to PheTyr (Figure 2).