Do qPCR Primers Need to Span Introns?

Do qPCR primers need to span introns? Here's the conclusion first:
If genomic DNA (gDNA) is removed during RNA extraction (e.g., using DNase I) or during reverse transcription, and only RNA remains, then designing primers that do not span introns typically has little impact.
However, if gDNA is not removed during RNA extraction, the resulting RNA sample will contain residual genomic DNA. In such cases, if the primers do not span introns, the primers may amplify DNA fragments in addition to cDNA, leading to falsely elevated expression levels of mRNA.

Why Does Spanning an Intron Help Avoid DNA Amplification?

To understand this, we first need to recognize the structural difference between DNA and mRNA. As shown in the figure below, when residual DNA is present, primers spanning an intron will bind to regions separated by a large intronic sequence in the genomic DNA.
Due to the characteristics of qPCR enzymes (which generally amplify fragments shorter than 500 bp), long genomic templates with introns are less likely to be amplified. Meanwhile, mRNA, which lacks introns, allows efficient amplification of the target fragment.

 Figure 1: Schematic diagram of DNA transcribed into mRNA

 To avoid amplifying genomic DNA during primer design, first examine the gene structure and choose a sufficiently long intron. Then, design forward and reverse primers on the exons flanking this intron.

In real-time PCR, the desired amplicon is typically short (100–300 bp), and PCR conditions are optimized for short fragments. Fragments exceeding 500 bp are difficult to amplify. If the intron between primers is long, genomic DNA amplification becomes inefficient or fails entirely.

 Figure 2: Primers spanning introns prevent genomic DNA amplification

What If the Intron Is Short?

If the intron is short, you can design one primer at the exon-exon junction, ensuring it spans the intron splice site. This also prevents genomic DNA amplification.
However, this method is not suitable for:

Genes with only a single exon

Organisms without introns

Species whose genome has not been annotated

 Figure 3: Primer design strategy for short introns

 Primer Design Tools

Design with sequence input: https://www.primer3plus.com/

Design using gene name: https://pga.mgh.harvard.edu/primerbank/

 Key Considerations for Primer Design

Optimal primer length: 18–30 nucleotides

Ideal melting temperature (Tm): 65°C to 75°C, with a Tm difference ≤ 5°C between forward and reverse primers

If Tm is too low, try increasing the GC content or slightly extending the primer length

Target GC content: 40–60%; primers should ideally end in C or G at the 3′ end to enhance binding

Add 3–4 extra nucleotides at the 5′ end before any restriction sites to improve enzyme efficiency

Avoid regions with secondary structures, and aim for balanced distribution of AT-rich and GC-rich regions

Avoid:

• Runs of 4 or more identical nucleotides or di-nucleotide repeats (e.g., ACCCC or ATATATAT)
• Self-complementarity within a primer (more than 3 bases)
• Complementarity between forward and reverse primers
  These issues can lead to primer-dimers or self-annealing, reducing specificity

For cloning, column purification is recommended as a minimum purification standard

For mutagenesis, place mismatched bases toward the center of the primer

For PCR primers used in Invitrogen TOPO cloning, do not phosphorylate the primers

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