Exercise 1: A Primer primer

In this section we will look at how primers are represented in Geneious.

Primers are a special file type in Geneious. A primer file will have a green arrow icon in the Geneious Document Table.

Select the T7 Promoter primer file provided with this tutorial to view it.

You will see that the primer comprises a DNA sequence with an associated primer_bind "binding region" annotation. This primer_bind annotation represents the region expected to bind to the primer's complementary target. Various metadata, including %G+C, melting temperature (Tm), Hairpin Tm and self-dimer Tm, are calculated and stored with the annotation when the primer is created.

Geneious uses Primer3 to calculate the Tm values. Note that Primer3 cannot calculate Tm statistics for primers with binding regions longer than 35 nucleotides.

The "binding region" defines the sequence considered when testing for a complementary match to a target sequence.

If you hover your pointer over the green Binding Region annotation a yellow Tooltip will appear and show the primer_bind metadata.


Primers may also include a 5' extension. This extension region is not covered by the primer_bind annotation as the extension region is not expected to participate in "first-round" annealing to a target sequence.

Select the second example primer sequence xynB R primer to view a primer with an extension comprising a 5'-PolyA sequence and a HindIII restriction site.


In Geneious various operations can add primers to target sequences as annotations, for example, using the tool Primers → Test with Saved Primers (covered in detail in section Exercise 6).

When a primer annotation is added to a sequence the primer_bind portion of the primer will be colored Dark Green if it binds in the forward direction (i.e. it complements the reverse strand), or light green if it binds in the reverse direction. If the primer has an extension then it will be depicted as a raised lightly-shaded non-binding region. If the extension has defined features then at higher magnifications these will be visible.

Select the DTU76545 sequence, zoom in on position 1140 and you will see binding position of the xynB R primer reverse primer annotated on the sequence. You can see the extension comprises a 5'-polyA region and a HindIII restriction site.

You will see that when annotated on a target sequence, a reverse primer annotation points right to left as it corresponds to the complementary strand of the target. As a consequence the extension sequence also reads from right (5') to left (3').



Use the link below to jump to Exercise 2.

Exercise 1: A Primer primer
Exercise 2: Manual entry of primers
Exercise 3: Importing tables of primers
Exercise 4: Manual design of primers
Exercise 5: Design of PCR primer pairs
Exercise 6: Testing primers against a target
Exercise 7: Degenerate primer design

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