Ignore Pseudogene

When activated, this option allow the filtering of Blast hits mapping to pseudogenes.

Word-size

This parameter represent the exact sequence match, as start region for the final alignment. Changing the initial word-size can help to find more, but less accurate hits; or to limit the results to almost perfect hits.

• Decreasing the word-size will increase the number of detected homologous sequences, but hits can include alignments of higher fragmentation due to gaps and substitutions.
• Increasing the word-size will give less hits as it requires a longer continuous regions of exact match. If this value is chosen to be almost the size of the query, BLAST will search for almost exact matches.

We recommend keeping the word-size below half the length of the primer size.

Primer Size

Define the minimal, maximal and optimal size of the primer sequences.

Primer melting temperature

Define the minimal, maximal and optimal melting temperature of the primer sequences.

Temperature delta penalty

Apply a minor penalty to an assay when the difference in melting temperature between the two primers is higher than this threshold.

Intron spanning assay only

When activated, only the assays overlapping an exon-exon junction will be reported. Deactivating this option will increasing the processing time of a query.

How many assay to display per transcript

When set to 0, every available assay is reported. Changing this parameter limit the number of assay reported per transcript.

Amplicon size

Define the minimal and maximal size of the PCR amplicon designed by Primer3. Smaller amplicon are favored over larger amplicon.