Making a Restriction Site Nonfunctional using PCR

If there is a enzyme recognition site in the middle of the gene that can disrupt the GGS, the following method shows how to change a single nucleotide base while maintaining amino acid identity, without altering codon usage.

Figure1: Media:PCRMutationdesign.pptx

Figure 1. Recognition site is marked in red. Two rounds of PCR produce two full length, linear DNA (Green and purple) without this site in plasmid pSB1A2. These products contain the same nucleotide sequence; however, one contains the mutations (curved line) at the 5’ end and the other has these mutations at the 3’ end. B. When strands from different PCR reactions anneal, a circular product is formed. Strands from the same PCR reaction cannot form a plasmid (Mary Gearing)

One linear PCR product contained the mutations at the 5’ end and the other contained the mutations at the 3’ end. Boil the combined products together as in standard oligonucleotide assembly and cool to allow annealing of strands. Only pairs that contain one strand from each PCR reaction can assemble into a circle to produce a plasmid.