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 firstname.lastname@example.org and let us know what you think of this new feature or provide any other comments or suggestions for improving NCBI BLAST products.
We have made some recent improvements to the BLAST+ applications that take full advantage of the version 5 BLAST databases (BLASTDBv5), which include built in taxonomic information for sequences and no longer rely on the integer sequence identifiers (gi numbers).
With the latest version of BLAST, you can now:
On Wednesday, November 1, 2017, we will present a webinar on GDV, NCBI’s full-featured genome browser. In this webinar, you’ll learn how to explore and analyze sequences and annotations for eukaryotic RefSeq genome assemblies. We’ll show you how to:
- Search across the entire assembly for genes, products and other markers or jump to a specific position or range
- Display any of seven preselected track sets highlighting various aspects of the assembly or create and load your own custom track sets from your NCBI account.
- Load and display submitted alignment data from NCBI’s GEO or SRA.
- Upload your own annotation and variant data
- Display BLAST or Primer-BLAST results on the assembly in the browser.
Date and time: Wednesday, November 1, 2017 12:00-12:30PM EDT
After registering, you will receive a confirmation email with information about attending the webinar. After the live presentation, the webinar will be uploaded to the NCBI YouTube channel. You can learn about future webinars on the Webinars and Courses page.
NCBI will discontinue both the NCBI Genomes (chromosome) and the Human ALU repeat elements (alu_repeats) BLAST databases in October 2017.
Better alternatives to NCBI Genomes (chromosome)
The existing NCBI Genomes (chromosome) database does not offer complete and non-redundant coverage of genome data. The newly added NCBI RefSeq Genomes Database (refseq_genomes) and the RefSeq Representative Genomes Database (refseq_representative_genomes) are more useful alternatives to the chromosome database. You can select these databases from the database pull-down list on any general BLAST form that searches a nucleotide database (blastn, tblastn).
Figure 1. The nucleotide-nucleotide BLAST database menu with the recommended (RefSeq Genome and Representative genomes) and deprecated (NCBI genomes (chromosomes) and Human ALU repeats) databases highlighted.
NCBI is retiring the e-PCR tool effective immediately. The good news is that an existing tool, Primer-BLAST, fills in nicely for the functions of both Forward and Reverse e-PCR, and has the additional benefit of de novo primer design.
A common task facing geneticists is to assay for sequence changes at particular locations in genes. These assays are often looking for changes in the coding exon of genes, and the target sequences are typically amplified using PCR from genomic DNA using a pair of specific primers. In this article, we will show you how to use NCBI Reference Sequences and Primer-BLAST, NCBI’s primer designer and specificity checker, to design a pair of primers that will amplify a single exon (exon 15) of the human breast cancer 1 (BRCA1) gene.
Here are the steps to follow to design primers to amplify exon 15 from human BRCA1: