Date: Fri, 29 Mar 2024 05:20:34 +0700 (NOVT)
Message-ID: <1902612797.88152.1711664434114@ugene.net>
Subject: Exported From Confluence
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This feature allows to search for primer pairs that span introns=
on the genomic sequence or exon junctions on the mRNA sequence.
Note that RT-PCR design is only available for mRNA/cDNA sequences with annota=
ted exons. There are several ways to obtain the cDNA for a corresponding DN=
A sequence.
- From NCBI or ENSEMBL database.
For example, one can download =
the TMPRSS2 transcript variant 1 from NCBI Genban=
k using identifier NM_001135099.1.
&nbs=
p;This can be also done from UGENE using option Access remote database or Search NCBI Genbank=
a>.
- Align the genomic and cDNA sequences using spliced aligner.
For this option one =
must have both genomic and cDNA sequences.
In UGENE the spliced alignme=
nt can be performed using the Spidey tool.
To run the alignment open the genomic sequ=
ence and select action Align Align to mRNA sequence.
The generated exon annotations can b=
e then exported using action Export Export sequence of selected annotations
To design primers for your mRNA sequence and go to the RT-PCR tab of the=
Primer Designer dilaog:
=20
=20
=
=20
=20
The following parameters are available:
Exon annotation name
To detect exon boundaries UGENE searches =
for exonic annotations. This option allows to set custom name for annotatio=
ns denoting exons. Default value is "exon"
Minimum exon junction overlap size&nb=
sp;
If checked, then only the pairs with at l=
east one of the primers overlapping exon junction in the mRNA sequence will=
be selected.
At 5' side (bp)
Minimum overlap size on the 5' side of th=
e exon junction. Default is 5 bp.
At 3' side (bp)
Minimum overlap size on the 3' side of th=
e exon junction. Default is 5 bp.
Exon range
This option allows to limit the seq=
uence region, where the primers are searched for. For example, setting valu=
e "3-5" will limit the search to a sequence region consisting of exons 3,4,=
5 of the transcript, as defined by the order in the sequence. Default value=
is an empty string, which means that there are no limitations.
Span at least one intron
This option makes sure that primer produc=
t should span an intron on the genomic sequence i.e the forward and reverse=
primers must be located in different exons. The option is enabled by defau=
lt.
Max numbers of pairs to query
The algorithm applied in RT-PCR primer de=
sign first searches for all available primers in a given sequence. Then it =
filters the detected pairs to make sure that they satisfy the selected conf=
iguration. This option allows to set the maximum number of pairs for the in=
itial search query. Larger number will result in increased sensitivity, but=
also in a longer running time. Default value is 1000.
Important: using the RT-PCR primer des=
ign tab will reset the values set in the Exlcuded regions and =
Targets of the Main configuration tab. Additionally i=
f the Exon range option is set, the defined sequence region will b=
e ignored.
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