Primer design in CLC tutorial
Go to parent Exercises for the Cloning tutorial
We will use the CLC Main Workbench to design primers for checking if a gene is successfully inserted in a vector. In this case specificity is not a big issue: the chance that a primer will bind another part of the (small) plasmid is very small. We will use the pcDNA3-atp8a1 sequence from the Cloning folder in the Example Data. This is the pcDNA3 vector with the atp8a1 gene inserted. We wish to design primers that allow us to generate a PCR product covering the insertion point of the gene to check if the gene is inserted where we think it is.
Double click the name of the vector, pcDNA3-atp8a1, to visualize it.
Click the Primer Designer button in the bottom menu:
We want to design primers with Tm=60°C and we want to check if the atp8a1 gene is inserted where we think it is. This means we want the forward primer upstream of the gene and the reverse primer inside the gene. If the gene is not inserted we will not get a PCR-product since the reverse primer cannot bind. If the gene is inserted elsewhere we will either see no PCR product (if both primers are too far apart) or a PCR product with a different size. In case we’re not sure about the length of the product, we can always sequence the PCR product to check the exact insertion site.
| Specify a region for the forward primer. |
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We want the forward primer in a region upstream of the atp8a1 gene, so somewhere between position 700 and 975 (start of atp8a1 gene).
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This will add an annotation to this region, Forward primer region in grey. Once you have added this annotation the Workbench will automatically search for primers using the default parameters. As a result, you see 5 rows of red and green dots representing the primer suggestions. Each row represents a different primer length - 18bp through to 22 bp, hence 5 rows. Red dots represent primer that do not meet the requirements defined by the prrimer parameters, green dots represent primers that do.
| How to change the parameters for the primer search ? |
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| On the right panel go to the Primer parameters section. Here you can specify the length and Tm of the primer (red). You can see more advanced parameters by expanding Advanced parameters (green):
 The advanced parameters specify the GC content of the primer and how tolerable you are towards the formation of hairpins (secondary structure) and binding of the primer to itself (self (end) annealing). The scores for self (end) annealing and secondary structure represent the number of hydrogen bonds involved in the binding. GC content is not that crucial but hairpins and primer dimers can mess up your PCR completely. Therefore I decrease the threshold of these 3 thresholds gradually until I have just a few green dots left.  |
As said before, each row consists of a number of dots, representing the starting point of a possible primer.
| Examine the primer suggestions and select a primer |
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| On the right panel go to the Primer parameters section. Here you can specify the length and Tm of the primer (red). You can see more advanced parameters by expanding Advanced parameters (green):
The advanced parameters specify the GC content of the primer and how tolerable you are towards the formation of hairpins (secondary structure) and binding of the primer to itself (self (end) annealing). GC content is not that crucial but hairpins and primer dimers can mess up your PCR completely. Therefore I decrease the threshold of these 3 thresholds gradually until I have just a few green dots left. Click the green dot that is the closest upstream atp8a1 to view the characteristics and the location of this primer:
 Now click the red dot right after the green dot you have just clicked. We set the minimum Tm at 58°C. This is the reason why the primer represented by this dot with a Tm of 57.8 does not meet the requirements and is colored red. There is an asterisk (*) before the Tm. This indicates that this criterium is not met by the primer.
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Warning: a dot represents the start of a primer so you must look for individual green dots: they represent primers that meet the requirements.
You do not need to look for 18 consecutive green dots: one dot represents a complete primer.
It is possible to add sequences e.g. restriction sites to the primers as you can do in SnapGene. For an example see the section on Restriction cloning in CLC.
Exercise: design the reverse primer
The reverse primer needs to be located in the CDS with a similar Tm as the forward primer. Try to find a reverse primer without peeking at the solution.
| Design the reverse primer. |
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 Set the Primer parameters to the same thresholds as you used for the forward primer.  Click the Calculate button to set the parameters for the primer pair:  You want two primers with very similar annealing temperatures so that they both bind well to the target during the annealing step of the PCR cycles. Additionally, you do not want the primers to bind each other and form dimers.  Clicking Calculate generates a table containing the ideal primer pairs in the regions that you have chosen and according to the criteria that you have set (for individual primers and for primer pairs). Primers pairs are ordered by a score (red) that reflects how well the pair satisfies the set criteria, so clicking the first row of the table highlights the highest scoring primer pair on the sequence:  The table also shows that the Tms of these primers are very similar (green) and they are not likely to bind each other (blue).
 As you can see both primers correspond to green dots meaning that they satisfy the criteria set for individual primers. |
| What does the score of the primer pair mean ? |
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The Workbench uses a proprietary algorithm to score primer pairs so we cannot give you much information except for what is mentioned in the manual:
CLC Main Workbench employs a proprietary algorithm to rank primer pairs. The algorithm considers both the parameters related to individual primers, such as the secondary structure score, and parameters related to primer pairs such as the pair-annealing score. The ideal score for a pair is 100 and pairs are ranked in descending order. Each parameter is assigned an ideal value and a tolerance. Consider for example the self-annealing score: the ideal value is 0 and the tolerance corresponds to the maximum value specified in the side panel. The negative contribution to the final score is determined by how much the parameter deviates from the ideal value and is scaled by the specified tolerance: contribution = (ideal - actual value)/tolerance Thus a large deviation from the ideal value and a small tolerance will give a large negative contribution in the final score and a small deviation from the ideal and a high tolerance will give a small negative contribution in the final score. |
The first primer pair seems very well, we are going to settle for this pair.
| How to save the primer pair for your experiment ? |
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| <p>Right click the row containing the primer pair you want to use and select Save primers:
 You need to select the folder you want to save the primers in and click Ok:
 In the navigation area you can now check if the primers were effectively saved:
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| How to keep the primer pair annotated on the sequence ? |
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If you paid close attention during the previous question you should already know: Right click the row containing the primer pair you want to use and select Mark Primer Annotation on Sequence:
 In the sequence view area you can see that the primers are annotated:
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As you can see in CLC you can define many criteria that the primers have to satisfy, allowing you to design very efficient pairs of primers.
However, CLC does not allow you to define criteria for the specificity of the primers as Primer-BLAST does. At this point in the primer design process there are no guarantees that the primers are specifically targeting the region you want to amplify and not any other region. In this example, the targeted region is located in a small plasmid, so it is very unlikely that the primers will bind to other regions in the plasmid. The longer the sequence, the higher the chance that primers of around 20nt are able bind to multiple regions in the sequence. So if you want to design primers for amplifying a region from the genome (or the transcriptome) you have to check the specificity of the primers to avoid decreasing the efficiency of the primers and generating aspecific PCR products.
