Purpose of this server

Designing oligos for PCR analysis is a common task that biologists face. Such oligos must be carefully chosen according to multiple caracteristics, such as hybridization specificity, melting temperature and amplicon length. In some cases, such as when investigating the presence of proteins on large genomic regions by chromatin immunoprecipitation experiments, multiple primer pairs must be designed, which is a time-consuming and repetitive task.

This server takes away the repetitiveness of designing multiple oligos at one or multiple given genomic loci by automating the process, relying on common tools that biologists use (manually) to do this task. In essence, the sequences are split in multiple subregions according to user specifications, Primer3 is used to identify candidate primer pairs and BlastN is used to control for oligo specificity within the chosen organism.

A typical use scenario

Suppose you want to design primers at every 75 base pairs of the sigH gene of Mycobacterium tuberculosis and that your amplicons should be 100-150 base pairs long. Clearly there will be some overlap in those amplicons. This scenario is one of the demonstration input provided on the home page. Try it! You may view its result by clicking HERE.

Loading the parameters

  1. Enter the sigH DNA sequence;
  2. Enter a job name (Optional); This is for your reference only and does not affect any computation.
  3. Specify the acceptable minimum and maximum melting temperature for your experiments; Here, the default 60-63 degrees is fine;
  4. Specify the acceptable minimum and maximum amplicon sizes for your experiments; Here, the default of 100-150 base pairs is fine;
  5. Specify the second tiling type option (base on the tiling distance), with a distance of 50 base pairs;
  6. Select a genome as the target organism (Bacteria-Mycobacterium_tuberculosis_H37Rv); If you have trouble finding it, use the filtering box below.
  7. You may override the default specificity checking parameters at this time;
  8. Provide an email adress if you want to receive notification of the result (Optional)
  9. Press submit!

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Interpreting the result

A PCRTiler result page is divided in the following sections:
  1. General information about the primer design job;
  2. A list of the best primer pair found for each sub-region. At the bottom of this section, you may download an oligo list in various formats. You may view supplementary information about this region by clicking on the "Details button". This includes a list of alternative primer pairs, the raw Primer3 output, and the raw BLAST result for each oligo;
  3. A visual representation of the positions of the designed primer pairs;
  4. A textual representation of the designed oligo positions, suitable for programs such as Genamics Expression;
  5. Data retention statement. You have the option of deleting your job result from the server.

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