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:
1. Search BRCA1 in the NCBI Nucleotide system.
3. Click the “Highlight Sequence Features” in the right-hand column of the sequence record to activate feature highlighting. You will see the coding sequence (CDS) feature of the gene highlighted.
4. Change the “Feature” pull-down list at the bottom left of the sequence display from “CDS” to “exon” and then navigate to exon 15.
5. Follow the FASTA link to display the highlighted exon as a separate view. Then follow the link in the right-hand column of the sequence display to “Pick Primers“.
6. Edit the primer ranges in Primer-BLAST so that the forward and reverse primers will bind upstream and downstream of the exon. For example, set the forward primer range from 146646 to 146746 and the reverse primer from 147056 to 147156. This will provide sufficient upstream and downstream sequence for Primer-BLAST to find acceptable binding sites.
7. We want these primers to amplify only the target region from the human genome sequence. Set the database for Primer-BLAST to perform a specificity check to “Genome (reference assembly from selected organisms)” and leave the Organism limit set to human.
8. Run the search with these settings by clicking the “Get Primers” button. An intermediate page appears that identifies a match to the chromosome 17 sequence (NC_000017.11). Check the box next to the accession to confirm that this is an allowed target and click the “Submit” button.
The results shown below in Figure 10 provide three candidate primer pairs for exon 15 of BRCA1.
The above procedure works well for designing exon-specific primers for any of the human genes with a RefSeqGene entry – more than 5,400 genes at present. For other genes, the Gene Sensor provides direct access to the Gene record and the Gene Table report available from the “Display Settings”.
The Gene Table provides the coordinates for and links to the exons on the corresponding NCBI Reference Sequence genomic record. You can then use the region as a template sequence in Primer-BLAST, just as with the RefSeqGene record.
Using the Gene Sensor, genomic Reference Sequences, and Primer-BLAST, you should be able to design primers that will amplify any region of interest for a gene from the human genome.