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Mutagenesis

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How do I design primers for site-directed mutagenesis?

Create Mutagenic Primers

In this example we design primers and simulate mutagenesis to change the Rab5 CDS at translated position 34 from a Serine to an Asparagine (S34N). We will change the codon "TCA" (S) to "AAC" (N). See Show Codon Frequencies to learn how to view synonymous codon usage frequencies for a host organism.

Annotate the Mutagenesis Region

Optional: It may be helpful to add a feature marking the position you wish to mutate.

Open a target circular plasmid sequence and identify the codon/site you want to change.

To add a feature, switch to Sequence view, then:

  1. Select the codon (three nucleotides) to be mutated and click Features → Add Feature....
  2. Give the feature an appropriate name.
  3. Set the "Type" to misc_feature.
  4. Optional: Add a note describing the feature.

Click OK to create the feature.

Identify the Primer Region

About 12-18 bases† of a mutagenic primer should anneal to the template on each side of the mutagenesis site.

  1. Click adjacent and upstream of the mutagenesis site, then drag to create a selection that has the desired length and Tm. Take note of the left-most nucleotide position.
  2. Click adjacent and downstream of the mutagenesis site, then drag to create a selection that has the desired length and Tm. Take note of the right-most nucleotide position.

† The length of the flanking sequence will be dependent on the %G+C content  of the target region, and the Tm of the mismatched mutagenic primers when bound to the target. Aim for a mismatched Tm suitable for PCR.

Select the corresponding region, then click menu Primers → Add Primer....

Chose to add the new primer to the "Top Strand".

Create the Forward Mutagenic Primer

In the "Add primer" dialog:

  1. Set a name for the top-strand mutagenic primer.
  2. In the primer sequence field, select the three nucleotides that correspond to the codon to be mutated.
  3. In the Codon section that appears, use the dropdown menu to select the replacement amino acid, and select the preferred synonymous codon.
  4. Click Insert to replace the selection with the new codon.
  1. The modification will be marked red in the primer field, hover over yellow arrow to see the codon translation.
  2. The binding mismatch caused by the codon change will be shown in the Sequence view.
  3. The calculated Tm for the mismatched primer is shown above the Sequence view †.

Click Add Primer to Template to create the primer and add it to your target sequence.

† The calculated Tm of this primer for the first round of PCR is 62°C. We would use this primer in a PCR with an annealing temperature of around 55-57°C.

Create the Reverse Primer

To create a complementary bottom-strand primer, select the primer in Map or Sequence view and click Primers → Duplicate Primer....

In The "Duplicate Primer" window:

  1. Add a Name for the new primer.
  2. Click Reverse complement to switch the primer to the bottom strand.
  3. The new primer binding position and orientation will be shown on the Sequence view.
  4. Hover over the marked changed nucleotides to confirm the codon translation.

Click OK to add the primer to the sequence.

Click File → Save to permanently add the new primers to the target sequence.

Perform Mutagenesis

Select a Mutagenic Primer

Select either a top-strand or a bottom-strand mutagenic primer, the click Actions → Mutagenesis....

The Mutagenesis tool assumes the use of a pair of perfectly matching complementary primers. However, you only need to select one of the two primers to use the tool.

Edit the Plasmid Name

  1. Confirm the appropriate mutagenic primer is selected.
  2. Confirm the changes via the summary displayed in the bottom panel.
  3. Enter an appropriate name for the new plasmid sequence.

Click Mutagenize to create the new plasmid.

View the New Plasmid and Confirm the Changes

Select a mutagenic primer in Map view and switch to Sequence view.

View the sequence and confirm the codon change.

Toggle on "Show Colors" to show "History Colors" and highlight the mutagenized codon.

Export the Primer Data

To export the mutagenic primers, switch to Primers view. Select the primers of interest, then click Primers → Export Selected Primer Data. See the "Export Primers" lesson for more details.

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