Note that the Primer3-derived Tm may differ slightly (∓1-3☌) from that given in the Selection Hint. Primer details, calculated by Primer3, are displayed in the Characteristics section of the dialog so that you can confirm the primer is appropriate for your intended application. In the Geneious Document table, select the file Primers as delimited text, then select and copy the three lines of 3-column tab-delimited text from the viewer pane: In this exercise we will import primers as tab delimited data (.tsv). Once you have confirmed the format the Import Sequences window will open. Geneious Prime will detect that you are importing tabular delimited data and ask you to confirm the import format. If you have primers in an Excel spreadsheet or a delimited text file then can simply Copy (CTRL-c or command-c) the column data from the spreadsheet, go to Geneious Prime and Paste (CTRL-v or command-v).
Geneious Prime will identify that you are importing tabular data and ask you to confirm the import format. Then either import via Geneious Prime menu File → From File…, or simply drag the delimited file into the Geneious Prime window.
If you have a collection of primers in an Excel spreadsheet then you can save the file in either tab-delimited (.tsv) or comma-delimited (.csv) format. Importing Primers from a delimited text document At a minimum the tabular data should have two columns, the first column for the primer name, the second for the primer sequence (5′-3′). You can easily import primer sequences from spreadsheets or delimited text documents. As a consequence the extension sequence also reads from right (5′) to left (3′). 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. You can see the extension comprises a 5′-polyA region and a HindIII restriction site. 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. If the extension has defined features then at higher magnifications these will be visible. If the primer has an extension then it will be depicted as a raised lightly-shaded non-binding region. it complements the reverse strand), or light green if it binds in the reverse direction. 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. In Geneious Prime various operations can add primers to target sequences as annotations, for example, using the tool Primers → Test with Saved Primers (covered in detail in Exercise 6). If you hover your pointer over the green Binding Region annotation a yellow Tooltip will appear and show the primer_bind metadata. The “binding region” defines the sequence considered when testing for a complementary match to a target sequence. Note that Primer3 cannot calculate Tm statistics for primers with binding regions longer than 35 nucleotides. Geneious uses Primer3 to calculate the Tm values. 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. This primer_bind annotation represents the region expected to bind to the primer’s complementary target. You will see that the primer comprises a DNA sequence with an associated primer_bind “binding region” annotation. Select the T7 Promoter primer file provided with this tutorial to view it. A primer file will have a green arrow icon in the Geneious Document Table.
Primers are a special file type in Geneious Prime. In this section we will look at how primers are represented in Geneious Prime.