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Primer Design Tutorial
Note: To complete the tutorial with the referenced data please download the tutorial above and install in Geneious Prime.
The design of PCR primers is relatively simple from a conceptual point of view: just search along a sequence and find short sub-sequences that fit certain criteria. However, the molecular biology of PCR is very complex and the design of primers is best accomplished with the aid of computer programs that can help decide what is a “good” and a “bad” primer.
Some general guidelines are:
- primers should ideally be 18-27 bp long
- have at least 40% G/C content
- anneal at a temperature in the range of 50 to 65 degrees
- avoid primers containing repetitions of the same nucleotide.
- avoid secondary structures (hairpins or primer dimer)
Usually higher annealing temperatures (Tm) are better (i.e. more specific for your desired target).
In addition, the forward and reverse primer should anneal at approximately the same temperature (allowing no more than 4 degrees of difference between them).
This Geneious PCR Primer design tutorial covers (1) basic design of PCR primers off a single sequence; (2) adding 5` extensions to primers; (3) testing existing PCR primers; and (4) design of degenerate primers off an alignment.
Exercise 1: Basic primer design
In this exercise you will design PCR primers to amplify part of the COX1 gene from the mammoth genome.
Click on the Mammuthus primigenius (woolly mammoth) mitochondrial genome sequence and locate the COX1 gene. If you are having trouble finding COX1 display the Annotations and Tracks tab to the right of the sequence viewer and type COX1 into the filter box.
Once you have the COX1 annotation selected, Click the Primers button from the menu options and select Design New Primers.
In this dialog box you can specify where you want to put your primers, what size PCR product you want to return, and characteristics such as size and melting temperature. Under Task select Generic, and check the Included Region box. This specifies where the primers will sit, so should be set to 5,331 to 6,878 – the base pairs that the COX1 gene spans. If there is a specific part of the gene you want to amplify in the PCR, set this under Target Region. For this example we don’t have a specific region of COX1 we want to amplify, but we want to ensure our PCR products are less than 300 bp long so that the primers will work on degraded DNA. So uncheck the Target Region box, and set product size to between 200 and 300 bp with an optimal size of 250 bp. Set Number of pairs to generate to 1.
The Tm Calculation section provides details on the formula used to calculate the melting point of oligos. You can leave this at the default settings.
Expand the Characteristics section. Here you can set the required properties of the primers, such as the length of the primer, the optimal melting temperature (Tm) and the penalties for hairpins and primer-dimers. For many applications the default settings will work fine, but you may need to adjust these if no suitable primers are found with the default settings, for instance if you have particularly GC or AT rich target sequence or the need for longer primers. For the primers we are designing here we can use the defaults for most of these options, with the exception of Max Tm Difference. Set this to 2 in order to restrict the Tm difference between F and R primers and ensure they will work in a single PCR reaction.
Your Design New Primers window should now look like this:
Click OK, and Geneious will now update the sequence with suitable primers displayed as annotations. Select the COX1 annotation and zoom in on it using the zoom button to the right of the sequence viewer. You should now be able to see primer annotations named with the base number they start at (if you cannot see them, make sure you have deleted COX1 from the filter box in the Annotations and Tracks tab). Click Save to save the primer annotations onto the sequence.
Place your mouse over the primer annotation and you will see a tooltip containing the characteristics of the PCR primer and product. You can see from this that these primers should amplify a product 250 bp long, and that the Tm for both primers is 60C.
Click on each primer annotation, then click Extract to extract the primers to your document table. Give the primers a meaningful name – e.g. “Mammoth COX1_F” and “Mammoth COX1_R”. They will then be available for use in other Geneious functions.
Exercise 2: Adding a 5` extension
A 5` extension is a short stretch of sequence at the 5` end of the primer that does not match the template. Examples of 5` extensions include restriction sites or cloning-specific sequences, where the aim is to produce a PCR product which can then be cloned into a vector, or sequencing barcodes and adaptors which enable PCR products to be sequenced using high-throughput sequencing. In Geneious, 5′ extensions are annotated separately so that this sequence will not be considered when testing primers against a target.
In this exercise, you will turn the primers we designed in Exercise 1 into fusion primers suitable for 454 Amplicon sequencing by adding a sequencing adaptor to their 5` ends. Select the forward primer you extracted in exercise 1. Click the Primers button from the menu options and select Add 5` extension. Click Bases. The sequencing adaptor we wish to add has two components – a 454 adaptor called “Primer A” (added to the forward primer) or “Primer B” (added to the reverse primer) and a linker sequence consisting of a 4 base pair tag. To add these, click Bases and paste the sequence of Primer A below into the bases box. Type Primer A next to Name and click OK.
Primer A: CGTATCGCCTCCCTCGCGCCA
Click the Bases box again, remove the Primer A sequence already there and type “TCAG” into the box. Type Linker next to Name and click OK. In the box you’ll see a visual representation of how the 5` extensions are arranged. It should look as in the screenshot below. You can reorder the extensions if you wish just by dragging and dropping them in this window.
Click OK and you will now see the primer displayed with Primer A and Linker sequences added to the 5` end. Click Save to update the primer. Repeat this process for the reverse primer, but this time add the Primer B sequence given below.
Primer B: CTATGCGCCTTGCCAGCCCGC
- For cloning applications, restriction sites and Gateway sites can also be added in the Add 5` extension dialog box.
- 5` extensions can also be added at the time the primer is designed under the Advanced options in the Design New Primers window
Exercise 3: Test Saved Primers
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: Designing degenerate primers
A degenerate primer contains a mix of bases at one or more sites. They are useful when you only have the protein sequence of your gene of interest so want to allow for the degeneracy in the genetic code, or when you want to isolate similar genes from a variety of species where the primer binding sites may not be identical.
The degeneracy value of a primer is the number of different primers that the primer sequence represents. For example, a primer which contains the nucleotide character N once (and no other ambiguities) has a degeneracy of 4 because N represents the four bases A,C,G and T. A primer that contains an N and an R has degeneracy 4 x 2 = 8 because R represents the two bases A and G. Because a degenerate primer is really a mix of different primers, only a fraction of the primer mix will work in an actual PCR. Thus it is best to limit degeneracy to under 100, as with higher values any one primer will become too diluted to work effectively, and non-specific target fragments may also be amplified. Degenerate bases at the 3` end of a primer should also be avoided.
In Geneious, you can design degenerate primers by using an alignment as the template. In this example, we will design primers to amplify an MHC class II gene, a highly polymorphic immune gene found in vertebrates.
Click on the MHC class II alignment. In the Display window next to the sequence viewer, check Highlighting and select Disagreements to Consensus.
From the alignment you can see that this gene contains polymorphic regions interspersed with conserved regions. We wish to design primers to sit in the relatively conserved regions at ends of the sequence to amplify across the polymorphic regions. We can do this by specifying the Target Region to amplify.
Select bases 41 to 264 in the alignment then click Primers → Design New Primers. These base numbers should show up in both the Target Region and Included Region options. As this is the region we wish to sequence, we don’t want our PCR primers within this region, so uncheck the Included Region box and check the Target Region box. Uncheck the product size range and optimal product size boxes if these are checked, and set Number of pairs to generate to 1. As the region from bases 1-40 where the forward primer will sit is still somewhat polymorphic, we will need a degenerate primer to bind to this region. Expand the Characteristics panel, check the Allow Degeneracy box and set the number to 300.
At the bottom of the window are options that allow you to control how primers are designed on alignments. For degenerate primers, choose to design them on the Consensus. Then click the Consensus options to set how variable a position should be to be made degenerate. In this window set the Threshold to 75%. This means that at any given site in the primer, the primer will match at least 75% of the sequences in the alignment. Thus if more than 75% of the sequences in an alignment have the same base at any given position, that position will not be degenerate in the primer. If you want a primer where every single variant base in every sequence is included in the primer, set the consensus threshold on 100%.
Your settings should now look as in the picture below. Click OK
You should find that one primer pair has been added to the consensus sequence. Mouse over the forward primer to bring up the tool tip. You’ll see that this primer has 4 degenerate sites specified by the letters Y, M, B, and R. These letters are IUPAC degeneracy codes which specify the mix of bases at that position. For a list of these codes see here. The degeneracy score for this primer is 24, which is a reasonable value for successful PCR. The second position in the primer is not degenerate as only 1 sequence in the alignment has a variant base (T) in this position, thus 5/6 (83%) of sequences match the primer, which is greater than the 75% threshold.
Click on each primer annotation and Extract them to your Document Table so they can be used in other functions.
This concludes the Primer Design tutorial.