This feature allows to span corresponding intron sequences search for primer pairs that span introns on the genomic sequence or junction sequences between exonsexon junctions on the mRNA sequence.
Note that this feature that RT-PCR design is only available for mRNA/cDNA sequences with annotated exons. There are several ways to obtain the cDNA for a corresponding DNA sequence.
- From NCBI or ENSEMBL database.
For example, one can download the TMPRSS2 transcript variant 1 from NCBI Genbank using identifier NM_001135099.1.
This can be also done from UGENE using option Access remote database or Search NCBI Genbank. - Align the genomic and cDNA sequences using spliced aligner.
For this option one has to must have both genomic and cDNA sequences.
In UGENE the spliced alignment can be performed using the Spidey tool.
To run the alignment open the genomic sequence and select action Align -> Align ‣ Align to mRNA sequence.
The generated exon annotation annotations can be then exported using action Export ‣ Export -> Export sequence of selected annotations
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This option makes sure that each detected primer pair product should span an intron in on the genomic sequence i.e the forward and reverse primers must be located in different exons. The option is enabled by default.
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The algorithm applied in RT-PCR primer design first searches for all available primers in a given sequence. Then it filters the detected pairs to make sure that they satisfy the selected configuration. This option allows to set the maximum number of pairs for the initial search query. Larger number will result in increased sensitivity, but also in a longer running time. Default value is 1000.
Note, that Important: using the RT-PCR primer design tab will reset the values set in the Exlcuded regions and Targets of the Main configuration tab. Additionally if the Exon range option is set, the defined sequence region will be ignored.
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