Virtual Cloning Tutorial - Cloning Primer Design

In Geneious R7 and above it is possible to create PCR primers by adding a primer annotation directly onto a sequence. This is especially useful for cloning applications as generally the primers must bind to a specified set of bases at the beginning and end of the gene to be cloned.

Open Human Proinsulin if it isn't already open. Select 18 bases from the beginning of the NcoI site into the proinsulin CDS (bases 54-71), then click Add Annotation. Make the annotation type primer_bind and give your primer an informative name (e.g. proinsulin_F). Once you have changed the type to primer_bind, primer-specific options and characteristics will show as in the screenshot below. These options allow you to preview the primer sequence, length and Tm etc and add a 5' extension if required.

You can see that this primer has a Tm of about 60°C, no hairpins and a low self dimer Tm so it is likely to be a good primer. Click OK to add this annotation to the sequence.

We don't want to include the stop codon our PCR product, so for the reverse primer select 18 bases in from base 385 (i.e. bases 368-385). Click Add annotation and choose primer_bind_reverse as the type. Give the primer a name (e.g. proinsulin_R). Check the primer characteristics, and you'll see that this primer has a Tm of 52.3°C, which is somewhat lower than the Tm of our forward primer. The Tm of the forward and reverse primer should be as close as possible, so try extending the primer site at the 5' end by changing the right-hand position of the binding site down to base 365. As you change the binding site position you'll see the primer sequence and characteristics automatically update. A primer spanning bases 365 to 385 has a Tm of 58.9°C, which is close to that of our forward primer.

The reverse primer also has to contain a NcoI site, and we can add this directly at this step by clicking the button next to Extension then clicking Restriction site and choosing NcoI as the enzyme. Because this is a reverse primer the NcoI site should also be reversed, so check the reverse box. Click OK to exit the primer annotation dialogs and add the primer annotation onto the sequence.

Click Save the document to save the primer annotations on the sequence, then Extract the two primers to your document table by selecting each one in the sequence view and clicking Extract.


Note: You can also design primers for cloning using the Design New Primers function under the Primers menu. To do this, select the region from the beginning of the NcoI binding site to just before the stop codon of the CDS (54 to 385), then select Primers→Design New Primers. Under Task select Cloning, as this will enable you to design primers to amplify a specific region. Make sure Included region contains the bases you selected, 54-385, and Number of pairs is set to 1. Click OK and you should see the same primers annotated onto the sequence as above.


Exercise 4: Introduce Cut Site using PCR
Exercise 5: Insert into Vector