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PCR Based Site Directed Mutagenesis

Polymerase Chain Reaction Site Mutagenesis

Learn about PCR based site directed mutagenesis.

(Refer to Diagram Below)

Begin first with a plasmid containing a gene with a TAC tyrosine codon that we want to alter to a TTC phenylalanine codon. In order to accomplish this we need to change the A-T pair (blue) in the original to a T-A pair. This plasmid was isolated from a normal strain of E.coli that methylates the As of GATC sequences. The methyl groups are indicated in yellow. The following steps are performed to accomplish this:

    • Heat the plasmid to separate its strands.
    • We then anneal mutagenic primers that contain the TTC codon, or its reverse complement, GAA. The altered base in each primer is indicated in pink.
    • We then perform a few rounds of PCR (about 8) with the mutagenic primers to amplify the plasmid with the altered codon. We use a heat stable DNA polymerase, such as Pfu polymerase, to minimize mistakes in copying the plasmid.
    • We then treat the DNA in the PCR reaction with DpnI to digest the methylated wild-type DNA. Since the PCR product was made in vitro, it is not methylated and is not cut. Lastly we transform E. coli cells with the treated DNA. In principle, only the mutated DNA survives to transform. This is checked by sequencing the plasmid DNA from several clones.

    Site Directed Mutagenesis

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