In silico PCR is used to calculate theoretical polymerase chain reaction (PCR) results using a given set of primers (probes) to amplify DNA sequences.
UGENE provides the In silico PCR feature only for nucleic sequences with Standard DNA and Extended DNA alphabets. To use it in UGENE open a DNA sequence and go to the In silico PCR tab of the Options Panel:
<center> <br> <img src="/wiki/download/attachments/65930776/In Silico PCR_0.png"/> </br> </center>
There are the following parameters:
Forward primer - forward primer. The primer length should be between 15 and 50 bases.
Reverse primer - on the opposite strand from the forward primer. The primer length should be between 15 and 50 bases.
Note: you should take in account, that algorithm calculates no more than 50 forward and 50 reverse primers, which means, that you can take 50x50=2500 products maximum.
Mismatches - mismatches limit.
3' perfect match - specify the number of nucleotides at the 3' end that must not have mismatches.
Maximum product size - the maximum size of the amplified sequence. Use ambiguous bases - search for ambiguous bases (as \"N\") if checked.
Extract annotations - specify the type of extracted annotations: Inner, All intersected or None.
Melting temperature - see the corresponding page for details.
Type two primers for running In Silico PCR. If the primers pair is invalid for running the PCR process then the warning is shown. Also, primers for the running In silico PCR can be chosen from a primer library. Click the following button to choose a primer from the primers library:
<center> <br> <img src="/wiki/download/attachments/13435073/Primers Library.png"/> </br> </center>
The following dialog will appear:
<center> <br> <img src="/wiki/download/attachments/16125631/In Silico PCR_2.png"/> </br> </center>
The table consists of the following columns: name, GC-content (%), Tm, Length (bp) and sequence. Select primer in the table and click the Choose button.
Click the Reverse-complement button for making a primer sequence reverse-complement:
<center> <br> <img src="/wiki/download/attachments/13435069/In Silico PCR_1.png"/> </br> </center>
Click Show primers details for seeing statistic details about primers.
When you run the process, the predicted PCR products appear in the products table.
There are three columns in the table:
Click the product for navigating to its region in the sequence.
Click the Extract product(s) button for exporting a product(s) in a file or use double click for that.
<center> <br> <img src="/wiki/download/attachments/13435069/In Silico PCR_2.png"/> </br> </center>