Primer-BLAST now has a “Primers common for a group of sequences” submission tab that allows you to design primers for a group of highly similar sequences. For example, you may want test for expression of any transcript of gene rather than a specific splice variant, so you want to design primers to cover all transcript variants. Or you may want to design primers that will amplify the same gene in closely related bacteria strains. To find primers for a group of related sequences, Primer-BLAST aligns the longest sequence to the rest to find common regions. It uses these to limit the locations of primers. The longest sequence is also used as the representative template sequence in the results. Figure 1 shows an example search for primers that will amplify all of the 15 splice variants for the human TP53 gene.
Figure 1. Primer-BLAST submission page and results for primers designed for the human TP53 transcripts. Top panel: The submission form with the “Primers common for a group of sequences” selected and the 15 RefSeq transcript accessions for TP53. Middle panel: The graphical results showing the longest sequence (NM_001126114.3) as the representative template, the locations of the primer pairs, and the alignment of the other template sequences. Bottom panel: An individual primer pair showing the locations on each of the template sequences.
Please try out this new feature and let us know what you think!
We’ve added a new feature (Max 3′ match), shown in Figure 1, to Primer-BLAST that limits the length of 3′ exon matches when designing exon-exon spanning primers. This makes it less likely that primers specifically designed to amplify transcripts will also amplify genomic DNA contamination in expression assays.
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.
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).
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
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).
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: