The Polymerase Chain Reaction (PCR) is a widely used molecular biology technique. When using it on a daily basis, very extense libraries can be generated to store the amplification products and the oligonucleotides (oligos or primers) used to produce them. Those databases contain important information to predict the efficiency of a PCR experiment, such as thermodynamic stability of primer-template binding, GC content, secondary structure formation, and other values that are all critical factors for amplification success.

On a high-throughput experiment, integrating algorithmic primer design tools enables you to streamline design, validation, and tracking of oligonucleotides directly within an automated workflow. In this article, we'll see how to work with primers using TeselaGen's platform.

TeselaGen allows you to manage your primers (referred to from now on as oligos). They are stored on the Oligos library inside the Molecular Biology Toolkit. Your oligos have the typical library view:

There are different ways to add more oligos to the library. The first one is to manually add them by clicking on the "New Oligo option". This will trigger a pop-up window, where you can name your oligo and indicate the sequence of it.

This is useful for manually-created oligos (remember that there is an automated oligo creation on every DNA Assembly Report). However, if you want to use TeselaGen's system to create oligos with your customized parameters, you can do it on the Open Vector Editor (OVE).

The OVE provides you with different tools to work with your DNA sequences. To create oligos on it, select the portiion of the sequence you want to amplify and right-click on it. Then, select Create > Create PCR primers for this region.

This will open a pop-up window where you can set all of your desired parameters, including product size, Tm, oligo size, GC content, and other advanced parameters.

Notice that, if the sequence you want to amplify is already recognized as a DNA part in your system, the window will give you the option to use the already existing part. On this window, you will also need to indicate which primer binding region you want to use.

For this example, let's select a small fragment of a sequence. After indicating our parameters, a pop-up window with the possible oligos will appear; here, we can select the oligo(s) that best fit our requirements. Notice that they show a penalty score; this is a measure of the quality of the oligo (the lower the penalty score, the better the oligo).

Once we've selected the oligo(s), they will appear on the OVE as annotations and will be there any time you open that specific sequence.

To edit the oligos, right-click on the annotation to see the available options:

When editing an oligo, you can change the bind start, bind end, and binding strand.

Inside an oligo's entry, you can see its binding sites on the corresponding section:

When clicking on "Finding More Binding Sites", you will see a list of all of the sites in your sequences that match the oligo. Take into account that this will screen all of your sequences on the project, so the longer your database, the more extense the analysis will be.

Once your oligos are all set, you can use them with tools, or to simulate a PCR.