Exercise 4: Manual Design of primers

Manual design of new primers

Geneious Prime includes a new feature for rapid manual creation of new primers.

If you select any region (100 nucleotides or less) of a DNA sequence then Geneious will display a floating Selection Hint that displays the length and Tm of the selected nucleotides. An Add Primer button sits to the right of the Selection Hint. You can select and drag to reposition the Selection Hint if it is in the way.

The Tm in the Selection Hint (RoughTm) is calculated dynamically as described in section 5.2.9 of the Geneious manual.

Once satisfied with the position and Tm of the selection then you can click the Add Primer button to open the Add Annotation dialog. The Add Annotation dialog will open with settings appropriate for creation of a new primer.

You can then give the primer a Name, set the primer Direction and, if required, add an Extension.

Primer details, calculated by Primer3, are displayed in the Characteristics section of the dialog so that you can confirm the primer is appropriate for your intended application.

Note that the Primer3-derived Tm may differ slightly (∓1-3°C) from that given in the Selection Hint. This is because Primer3 uses a more sophisticated algorithm that considers primer, nucleotide and buffer salt concentrations. You can click on the Tm Options link to set the component concentrations to match those to be used in your intended experiment.

Exercise:

In this exercise will design a new Forward primer for use in PCR with a pre-existing reverse primer.

The DTU76545 target sequence we will use contains the bacterium Dictyoglomus thermophilum xynB CDS. The primer we will create will amplify the region encoding the mature XynB gene product.

The new primer will be designed to include an extension that incorporates a NcoI restriction site into the PCR product. The NcoI site will be positioned to allow ligation of digested PCR product into the pET26B expression plasmid such that an in-frame fusion will be generated between the xynB CDS and a plasmid-encoded PelB signal peptide. We will also add a 5'-polyA spacer to ensure that NcoI enzyme will be able to cut the new NcoI site efficiently.

The binding region of our new primer should have a Tm of around 55°C to match the Tm of the xynB R primer Reverse primer that is already annotated onto the DTU76545 sequence.

1. Select the file DTU76545 and zoom in/scroll to around position 140 bp.

You will see a pink Signal peptide annotation that defines the boundary between the predicted signal peptide of XynB and the mature xylanase enzyme. We will design a primer that amplifies from the last codon (Ala) of the signal peptide.

2. Go to the Display tab in the viewer panel and turn on Translation. Make sure the Frame setting is By Annotation. This will ensure we see the translation of the xynB CDS.

3. Select the pET26B vector and zoom into position 5137 bp. You will see the NcoI site that we wish to use to generate an in-frame fusion with the PelB leader sequence. Turn on Translation by Annotation as described in step 2. You will see that the ATG within the NcoI site (ccATGg) encodes a Methionine (M) in the PelB CDS. The last "G" nucleotide in the NcoI corresponds to position 1 in the next codon. We will use this positioning to ensure we position the NcoI site in our new primer to create an in-frame fusion between PelB and the mature XynB.

4. Switch back to the target DTU76545 sequence. Click and drag from the second position of the last codon of the signal peptide (CT - Ala) to select a region that has a predicted Tm of around 55°C. Click on the Add Primer button to open the Add Annotation dialog.

5. In the Add Primer Dialog, give the primer the Name: xynB F primer, confirm the direction is set to Forward, check the Characteristics and confirm they are OK, then hit the Extension button to add a new extension.

6. Click the Add: Restriction Site button, choose NcoI from the dropdown list, then go OK.

7. Click the Add: Bases button, type "AAAA" into the Bases: field, give the addition the name "Poly-A" and set Type: to "Poly-A", then go OK OK.

8. Confirm the Poly-A is the left-most (5'-) extension "part", you can drag and drop the parts to reorder them if required.

9. Go OK to close the Primer Extension Dialog, then go OK again to close the Edit Annotation Dialog. You should now see a new primer annotation associated with the sequence. You can see that the "ATG" within the NcoI extension is in-frame with the xynB CDS. Click the Save button (or type CTRL-S or command-S) to Save.

10. In the last two steps of this exercise we will confirm new primer generates the desired PCR product. Select the DTU76545 file, then in the Geneious Toolbar go Primers → Extract PCR Product.... The tool will detect the primer annotations on the sequence and select them for you. Click OK to proceed, a new PCR product file will be created.

11. Select the new DTU76545 PCR Product and zoom in to view each end. You will see the primer extensions are now incorporated into the PCR product sequence. You can also confirm that the NcoI-derived ATG codon is in frame with the xynB CDS.

Use the link below to jump to Exercise 5.

Exercise 1: A Primer primer
Exercise 2: Manual entry of primers
Exercise 3: Importing tables of primers
Exercise 4: Manual design of primers
Exercise 5: Design of PCR primer pairs
Exercise 6: Testing primers against a target
Exercise 7: Degenerate primer design

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