Things you can do to maximize the chance of finding primers specific for your template.
- Use refseq accession or GI (rather than the raw DNA sequence) as template whenever possible. In addition, make sure you are using the latest version of a refseq entry (i.e., the accession with the highest version number or simply just use the accession without the version number as you will automatically get the latest version).
Even if you are only interested in part of the sequence (for example, a region on chromosome), you should still use the accession or GI but you do need to specify the range (use forward primer "From" field for your sequence start position and reverse primer "To" field for your sequence stop position). The reason is that an accession or GI carries accurate information about its identity which allows primer-blast to better distinguish between intended template and off-targets.
- Try "re-search for specific primers" feature on Primer-BLAST result page. When only non-specific primers are found, the result page offers the option to re-search for specific primers. For example, a splice variant that is labeled as an unintended target may not be expressed at all in the cells that a user works with and therefore this splice variant can be safely ignored. To use such feature, users can simply mark the checkbox in front of the unintended template and then click the Submit button on the result page.
- Try to use "User guided" option under the "Search mode" parameter.
- Choose a non-redundant database (i.e., Refseq mRNA, Refseq representative genome or Genomes for selected organisms (primary reference assembly only) database). Other databases like nr contain redundant entries which likely will interfere with the process of finding specific primers.
- Exclude predicted transcripts from database search if you are not concern about them (i.e., select the "Exclude predicted Refseq transcripts" option). The existence of these transcripts are based on automatic gene predictions and users need to decide whether to include these sequences in your primer specificity checking process. Including them will certainly make it more difficult or impossible to design specific primers since they likely will share high similarity with your intended transcripts from the same genes.
- Allow primers to amplify mRNA splice variants. This will make it much easier to find gene-specific primers if you do not need to distinguish between splice variants.
- Specify an organism for database search if you are only amplifying DNA from a specific organism. Searching all organisms will be much slower and off-target priming from other organisms are irrelevant.
- Lower the primer specificity requirements if you are only concerned with targets that have perfect or nearly perfect matches to your primers...By default, primer-blast uses stringent parameters that can detect targets having significant number of mismatches to primers (in addition to perfectly matched targets). See Primer specificity stringency under Primer Pair Specificity Checking Parameters for more details.
- Increase the value for the "Max primer pairs to screen" option.
Last modified: Thu Nov 19 17:46:22 EST 2020