In Silico PCR Overview
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/16125631/In Silico PCR.png"/> </br> </center>
There are the following parameters:
Forward primer - forward primer.
Reverse primer - on the opposite strand from the forward primer.
Mismatches - mismatches limit.
3' perfect match - specify the number of nucleotides at the 3' end that must not have mismatches.
Maximum product - maximum size of the amplified sequence.
Extract annotations - specify the type of extracted annotations: Inner, All intersected or None.
- Value Inner is selected by default. When this value is selected, the extracted PCR product contains annotations from the original sequence, located within the extracted region.
- Value All intersected specifies that all annotations of the original sequence that intersect the extracted region must be extracted as well.
- Value None specifies that annotations from the original sequence must not be extracted.
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:
- region of product in the sequence
- product length
- preferred annealing temperature
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>