This program integrates primer3 and OligoCalc functions to pick candidate primers for staining.
- Take RNA sequence as input
- Call Primer3 to find hybridization probe (internal oligo) candidates with 21 bases with melting temperature 45~65 (celcius)
- Pick candidates ending with “TA” or “AG”
- Generate DNA primer pairs (20+20) for valid candidates
- Use the OligoCalc algorithm to check the melting temperature and self-complementarity of primer pairs
- Output valid primer pairs
- Download the entire "primerfinder" project to local machine. Creat a folder named "primer3" under the project folder. Unzip the "primer3.zip" file and place all primer3 files directly under the primer3 folder.
- Open Windows command line
- Go to the project folder
- Save nucleotide sequence in a file in the input folder, such as “.\input\SCGB1A1.txt”
- Make sure Rscript.exe is in your system path
- Run the program by typing “Rscript primerfinder.R .\input\SCGB1A1.txt”
- The output will be saved in a “uidxxx” folder in output directory
| File | Description |
|---|---|
| primer3.in.txt | the generated input file for primer3 |
| primers.1.all.txt | all primer candidates returned by primer3 |
| primers.2.validending.txt | all primer candidates with valid ending |
| primers.3.pairs.txt | all primer pairs |
| primers.4.final.txt | valid primer pairs |
- Currently support Windows system.
- Currently only accept bases: A, C, T, G, U.
- The program is in the testing phase. Highly recommend double checking the properties of the final primers in OligoCalc (http://biotools.nubic.northwestern.edu/OligoCalc.html).
- The implementation of the self-complementarity checking algorithm is more stringent than the implementation in OligoCalc.
- The Windows executables of primer3 (libprimer3 release 2.3.6) were downloaded from http://primer3.sourceforge.net/.
- The primer3 execution results are consistent with this web version of primer3: http://primer3.ut.ee/.