Primer-BLAST, NCBI’s primer-designer and specificity-checker, now offers a way to help you with irrelevant off-target matches.
Sometimes Primer-BLAST can’t design specific primers for your target sequence because of similar non-target sequences in the database. In some cases, you may know that these non-target matches are not important your research and are safe to ignore. Examples may include tissue-specific splice variants, redundant entries, and predicted sequences. To help in these cases, you can now choose to allow certain off-target matches. This gives Primer-BLAST greater freedom in primer selection and a better chance of finding highly specific primers.
How to use it
When you get results with off-target sequences you want to allow Primer-BLAST to match, simply check the box next to the ones you want to allow and click the ‘Submit’ button at the top of your results to re-run the search.
For example, when designing primers for NDUFB8 transcript variant 1 (NM_005004.4), Primer-BLAST finds an unintended match to transcript variant 2 (NM_001284367.1). It also finds some potential matches to RHOT2 transcripts (Figure 1). Re-running the search after allowing the match to NDUFB8 transcript variant 2 provides specific primers that don’t have the potential cross-matches to the RHOT2 transcripts (Figure 2).
Figure 1. Primer-BLAST results showing matches to unintended templates including transcript variant 2 of NDUFB8 and weaker matches to unrelated transcripts from RHOT2. Checking the box for transcript variant 2 and clicking the Submit button finds more specific primers shown in Figure 2.
Figure 2. Specific primers found by allowing matches to the off-target NDUFB8 transcript variant 2. These new primers do not have the potential matches to RHOT2 transcripts that were in the initial search shown in Figure 1.
We welcome your feedback! Please write to us at blast-help@ncbi.nlm.nih.gov and let us know what you think of this new feature or provide any other comments or suggestions for improving NCBI BLAST products.
Figure 1. NCBI’s web-based structure 