13.1.9 Output from Primer Design

Once the task and options have been set, click the OK button to design the primers. A progress bar may appear for a short time while the process completes. When complete, primers and probes will be added as annotations on the sequences. The annotations will be labelled with the base number the primer starts at, followed by either F (forward primer), R (reverse primer), or P (probe). Primers will be coloured green and probes red.

Detailed information such as melting point, tendency to form primer-dimers and GC content can be seen by hovering the mouse over the primer annotation. The information will be presented in a popup box. Alternatively, double clicking on an annotation will display its details in the annotation editing dialog. Table 13.1 shows how the values in the Geneious primer annotation map to the original Primer3 values.

If you are designing primers off an alignment the primer will be designed on the consensus sequence by default. To design primers for every sequence in the alignment and have the primers annotated separately on each sequence or on a few selected sequences, choose Design primers on “Every Sequence”, or “Selected Sequences” in the alignment options at the bottom of the Design New Primers window. This option is only available in Geneious 8.1 onwards.

The best way to save a primer or DNA probe for further testing or use is to select the annotation for that primer and click the Extract button in the sequence viewer. This will generate a separate, short sequence document which just contains the primer sequence and the annotation (so it retains all the information on the primer). In the case of the reverse primer it will automatically be reverse complemented.

To delete primers that you don’t want, just select the primer annotation and click the Delete button. You will then be given the option to delete the pair of that primer at the same time.


PIC


Figure 13.4: Primer design output






Geneious Primer Characteristics Primer3 Web Interface Primer3 Command Line



%GC Primer GC% PRIMER_{LEFT,RIGHT}_GC_PERCENT



Tm Primer Tm PRIMER_{LEFT,RIGHT}_TM



Hairpin Max Self Complementarity (Any) PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_SELF_ANY



Primer-Dimer Max 3’ Self Complementarity PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_SELF_END



Monovalent Salt Concentration Concentration of monovalent cations PRIMER_SALT_CONC



Divalent Salt Concentration Concentration of divalent cations PRIMER_DIVALENT_CONC



DNTP Concentration Concentration of dNTPs PRIMER_DNTP_CONC



Sequence Seq PRIMER_{LEFT,RIGHT}_SEQUENCE



Product Size Product Size Ranges PRIMER_PRODUCT_SIZE



Pair Hairpin PAIR ANY COMPL PRIMER_PAIR_COMPL_ANY



Pair Primer-Dimer PAIR 3’ COMPL PRIMER_PAIR_COMPL_END



Pair Tm Diff Max Tm Difference PRIMER_PRODUCT_TM_OLIGO_TM_DIFF




Table 13.1: Geneious primer characteristics and their Primer3 counterparts