Human F9 Gene: Designing qPCR Primers using PrimerBLAST
The RefSeq entry NM_000133.3 contains the sequence of the human mRNA coding for coagulation factor F9. The gene contains 8 coding exons and gives rise to a transcript of 2780 bp encoding a protein of 461 amino acids. Next, we want to design primers to measure the expression of the F9 gene.
Go to the RefSeq record of this transcript to study its structure. When you scroll down to the features section you see that the CDS is located from position 40 to position 1415. Since RNA degradation starts at the 5’end of transcripts, we don’t want to pick primers at the 5’end. On the other hand, we don’t want to pick primers in the long 3’UTR either because it doesn’t contain any introns (the exons are all coding) and we want to design exon-exon junction or intron spanning primers. Let’s try to find exon-exon junction spanning primers between position 400 and 1600, with optimal anneal temperature = 60.
| Find primers that fulfill the above defined criteria |
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| Go to PrimerBLAST and fill in the form as follows: |
Exclude predicted sequences in the database to search in .
| Find primers that fulfill the above defined criteria |
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| Go to PrimerBLAST and fill in the remainder of the form as follows: |
The PrimerBLAST gives you a set of 10 primer pairs. Look at the detailed report of the first primer pair:
As you can see the primers are not specific: they can bind to various other targets albeit with lower affinity because of the mismatches . The best option seems to be primer pair 7, which binds to both F9 transcript variants and potentially to one unintended target, but as you can see the last nucleotide at the 3’ end of both primers are specific.