Question 14 of 20 Intermediate Concept
Why are RT-qPCR primers often designed across an exon-exon junction, and when is that not enough?
Short answer
A primer that spans a junction matches spliced cDNA but not the genomic locus, where an intron separates the two halves, so genomic DNA amplifies poorly or not at all. It is not enough when the gene has a processed pseudogene, has no introns, or when the junction is shared with isoforms you did not mean to measure.
Why
In genomic DNA the two exons are separated by an intron. A primer whose 3' end crosses the junction cannot bind there, and a primer pair on two different exons gives a much longer genomic product that often fails to amplify. That keeps the assay specific to RNA.
There are three common gaps. First, a processed pseudogene is a spliced copy of an mRNA reinserted into the genome. It already lacks the intron, so junction primers amplify it from DNA as happily as from cDNA.
Second, single-exon genes and many bacterial or organellar transcripts have no junction to use. Third, a short intron can still be amplified, giving a second product. Isoforms matter too.
A junction present in several splice variants measures their sum, while one unique to a variant measures that variant alone. Tools such as ExonSurfer align candidate primers against both the transcriptome and the genome for this reason, and still report possible off-target isoforms. For example, primers for a gene with a pseudogene on another chromosome can pass every design rule and still amplify DNA.
A no-RT control is what reveals that.
A strong answer covers
- Junction sequence exists in cDNA but is split by an intron in the genome
- Processed pseudogenes defeat junction design
- Single-exon genes and short introns are further gaps
- Junction choice decides which isoforms are measured
- Check primers against transcriptome and genome, then run no-RT controls
Common mistakes
- Dropping no-RT controls because the primers span a junction
- Ignoring that a junction shared by several isoforms reports their combined level
- Assuming primers on two exons are always safe even when the intron between them is short
The interviewer might ask next
How would you design an assay that measures only one splice variant of a gene?