Exercise 6: Testing primers against a target

There are two methods for testing primers against a target sequence. Both methods require that you already have the target sequence present in your Geneious data folder.

  1. Add Primers to Sequence - Use this tool when testing new primers that need to be added to your Geneious database

  2. Test with Saved primers - Use this tool when testing primers that are already present in your Geneious database

These two methods are covered in the exercises below.

Manual entry and testing of one or more primer sequences using "Add Primers to Sequence"

The Add Primers to Sequence command allows you to simultaneously create and test pre-existing primers against an target sequence. For example, you might want to use primers found in a publication against your target sequence.

When the Add Primers to Sequence tool is run the primers are added as annotations to the target sequence if a match is found. You also have the option to output corresponding stand-alone primer sequences. To use the Add Primers to Sequence tool, select your target sequence/s and go Primers → Add Primers to Sequence.

Exercise:

In this exercise we will enter and test a pair of "Universal" primers for the amplification of prokaryotic 16S SSU sequences. These primers have been used to amplify the 16S rDNA gene from many bacterial and archeal species. We will use a published E. coli 16S SSU gene extracted from a published genome as our target sequence.

Select the E. coli K12 16S SSU target sequence.

We will use the following two primers.

Go Primers → Add Primers to Sequence, enter the Name: and Sequence: of each primer, use the orange + button to add the second primer. Check the option to Allow 1 mismatch. Check the option to Extract primers to folder. Click OK to run the Add Primers tool.

The new primers will be added to the target if a match is found, and new primer files will be created for each sequence.


Using Test with Saved Primers

This tool allows you to test primers already in your database against target sequences to determine how PCR products may look, or to check against other sequences for possible non-specific amplification. To use this tool, select your target sequence/s and go Primers → Test with Saved Primers.

Exercise:

In this exercise we will test the mammoth COX1 primers we designed in Exercise 5 using the African Elephant COX1 gene as the target sequence.

Select the DQ316068 - Elephas maximus mitochondrion, complete genome. Locate and select the COX1 CDS, then go Primers → Test with Saved Primers. You can leave Source: set to Use current folder, as this tutorial folder (Primer design) will contain the primers you created in exercise 5. Alternatively, you use the Choose... button to define the specific primers you wish to test.

Check the options for Forward Primer, Reverse Primer and Pairs only. Make sure the option Region: is set to Find primers that bind inside the selected region.

As the primers we are testing were designed for a different species from our target sequence, it is unlikely they will be an exact match, but they should still work in a PCR reaction with 1 or 2 mismatches as long as these are not too close to the 3' end. Allow for this by checking Mismatches and setting the Allow mismatches in binding region to 2. Set the option No mismatches within to 3 bp of 3' end. Click OK and your settings should now look like this:

Click OK, and you will see your Mammoth primers annotated onto the COX1 gene on the Elephant sequence. Select the forward primer annotation and zoom in. The tooltip that pops up when you mouse over the primer annotation shows you where any mismatches with the target sequence are. You can see that this primer has only one mismatch at the 5' end. Now go to the reverse primer, and you'll see a single mismatch in the middle of the primer. These mismatches are unlikely to affect PCR, so these primers should be successful in amplifying elephant COX 1 sequences.

Click Save to save your primer annotations onto the sequences. Once you have annotated the primer sites, you can extract the PCR product sequence for use in downstream analyses. Select the DQ316068 - Elephas maximus mitochondrion, complete genome again and choose Primers → Extract PCR Product. Ensure the Mammoth COX1 primers are selected and click OK. Because these primers have a mismatch with the target you will be given the option of either extracting the target sequence or the primer sequence. During the PCR process the primer sequence will predominate, so choose Extract Primer Bases. You should now see the PCR product sequence in the Document Table.

Use the link below to jump to Exercise 7.

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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