Designing exon-specific primers for the human genome


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.

BRCA1 search in NCBI Nucleotide

Figure 1. Search for “BRCA1” in NCBI Nucleotide.

 

2. Follow the Genomic link in the Gene Sensor box at the top of the Nucleotide results to retrieve the RefSeqGene record (NG 005905) for the BRCA1 gene.

brca refseq record

Figure 2. The Gene Sensor box located at the top of the Nucleotide results contains a Genomic link. Click it to retrieve the RefSeqGene record for the BRCA1 gene.

 

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.

highlight sequence features

Figure 3. “Highlight Sequence Features” is the third link under the right-hand side column titled “Analyze this sequence”.

 

cds highlighted

Figure 4. The coding sequence (CDS) feature is 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.

exon display

Figure 5. After changing the feature from “CDS” to “exon” (bottom left of sequence display), the exon is now highlighted.

 

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“.

pick primers

Figure 6. The FASTA link shows the highlighted exon as a separate view. Next, click on “Pick Primers” (the third link in the right-hand side column titled “Analyze this sequence”).

 

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.

edit primer ranges

Figure 7. Edit the primer ranges in the fields on the right-hand side. Set the forward primer range from 146646 to 146746, and the reverse primer range from 147056 to 147156.

 

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.

specificity check

Figure 8. Set the database to “Genome (reference assembly from selected organisms)”. 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.

click submit button

Figure 9. This intermediate page shows a match to the chromosome 17 sequence. Check the box next to the accession number to confirm that this is an allowed target and click “Submit”.

 

The results shown below in Figure 10 provide three candidate primer pairs for exon 15 of BRCA1.

primer blast output

Figure 10. Primer-BLAST output showing candidate primer pairs for exon 15 of human 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”.

display settings gene table

Figure 11. The Gene Table option under “Format”.

 

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.

exon table

Figure 12. The gene table showing coordinates for and links to the exons on the corresponding NCBI Reference Sequence genomic 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.

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