Fruit Fly Tap Gene: Designing qPCR Primers using PrimerBLAST

The RefSeq entry NM_079400 contains the sequence of the D. melanogaster mRNA coding for tap, the target of Poxn. Tap encodes a bHLH protein expressed in larval chemosensory organs and involved in the response to sugar and salt. We wish to amplify the region encoding the Helix-loop-helix domain. In the sequence of the RefSeq record, the domain is located between position +577 and +745.

We want to design qPCR primers for measuring the expression level of the hlh domain using SYBR green. Remember that it is advised to design intron/exon-exon junction spanning primers for qPCR experiments that are based on fluorescent labels to detect/avoid amplification of contaminating genomic DNA.

Check in NCBIs Gene database if the hlh domain contains any introns: to know the location of the introns, you need the genomic sequence instead of the mRNA sequence.

  • Go to the NCBI RefSeq record.
  • In the right menu click the link to the Gene record
  • In the Genomic regions, transcripts and products section you can see that the gene contains no introns: the transcript is not chopped up into pieces when aligned to the genome. Click here for an example of a gene with introns.

Next, we will design primers to measure the expression of the hlh domain.

Go to Primer BLAST by using the link in the Refseq record
Go back to the RefSeq mRNA record. There, you can go directly to PrimerBLAST by clicking the Pick Primers link in the Analyze this sequence section of the right menu.

Since you want to measure the expression of the hlh domain you want primers that are located inside the domain.

Define the range of the sequence in which you want to design primers.
You have to specify the range as follows:
Define the primer parameters to comply with the rules of qPCR primer design: product size and Tm.
To comply with the rules for qPCR primer design, you have to change the settings for PCR product size and melting temperature:
The PrimerBLAST automatically decides to check primer specificity in the Drosophila (organism ID: 7227) RefSeq mRNA database which is exactly what you want. For the qPCR you are going to use RNA samples from fruitfly. This means that the primers will only come into contact with Drosophila mRNAs so you only have to check their specifity in this database. Make sure the last 2 nucleotides are completely specific.
You want to ensure that the 3’ end of the primers really is specific:

The PrimerBLAST gives you a set of 9 primer pairs that are specific (according to the criteria that you have specified) and that fulfill all other requirements that you have defined. Look at the detailed report of the first primer pair: All parameters are quite self-explanatory except for the Self complementary and Self 3’complementarity scores.

  • The first score represents the local alignment score when aligning a primer to itself. The scoring system gives 1.00 for a match, -1.00 for a mismatch. This means that the lower the score (the more mismatches), the less likely that the primer binds to itself.
  • The second score represents the global alignment score when aligning a primer to itself. Here again, the lower the score, the better.

The scores are followed by information on the specificity of the primer: alignments of the two primers to all target sequences from the database that match the criteria that you specified. In these alignments dots represent matching nucleotides while letters represent mismatches. A specific primer pair will have two alignments (one for each primer): both perfect alignments (all dots) to the sequence you want to amplify.