The Test with Saved Primers function allows to you test primers in your database against target and other sequences to determine how PCR products may look, or test for non-specific amplification. In this section we will test the mammoth COX1 primers we designed in the Exercise 1 on the African Elephant genome.
Select the elephant mitochondrial genome sequence. Select Primers → Test with Saved Primers, and ensure Test specific primers against the selected sequences is selected. Check the Forward Primer box and click Choose. The list that comes up contains all the primers in your database and you should be able to find the Mammoth COX1_F and Mammoth COX1_R primers you designed in Exercise 1. Select the Mammoth COX1_F primer and click Select. Now check the Reverse box and select the Mammoth COX1_R primer.
Uncheck the Target Region, Included Region, Product Size, and Optional Product Size boxes. As the primers we are testing are from 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 Maximum mismatches and setting the number to 2. Click on Mismatch options and check Annotate Mismatches and No mismatches within 3 bp of 3' end. Click OK and your settings should now look like this:
Click OK, and you will see the primers annotated on your 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 elephant mitochondrial genome sequence 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.
Exercise 4: Degenerate primer design
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