Question 12 of 20 Fundamentals Interpretation
Why do you run a melt curve after a SYBR Green qPCR, and how do you read it?
Short answer
Amplification curves cannot tell the intended product from primer dimers or off-target products, because the dye binds all of them. A melt curve heats the products while recording fluorescence, and one sharp peak at the expected melting temperature is evidence, though not proof, of a single specific product.
Why
As the temperature passes a product's melting temperature, its strands separate and the dye is released, so fluorescence drops sharply. Software plots the negative first derivative of fluorescence against temperature, which turns each product into a peak. A single tall peak at the temperature seen in a validated positive control is the expected pattern.
A second, lower-temperature peak, often strongest in the no-template control, usually means primer dimers, because short duplexes melt at lower temperatures. A shoulder or a split peak can mean an off-target product, or an amplicon with uneven GC content that melts in two blocks. For example, if samples peak at 84 C and the no-template control peaks at 74 C, the control signal is probably dimer rather than contamination.
MIQE 2.0 warns that two different products can share a melting temperature, just as two can share a size on a gel. One clean peak therefore supports specificity but does not identify the amplicon. Sequencing the product, or a gel during validation, settles identity.
A melt curve is not needed for probe assays, where the probe already provides sequence specificity.
A strong answer covers
- Dye signal cannot distinguish products during amplification
- Derivative plot turns each product into a peak
- Low-temperature peak in the no-template control suggests primer dimers
- A single peak supports but does not prove identity
- Confirm identity once by sequencing or a gel during validation
Common mistakes
- Treating one melt peak as proof that the product is the intended amplicon
- Calling any no-template control signal contamination without checking its melting temperature
- Comparing melt temperatures across instruments or buffers without a reference control
The interviewer might ask next
If samples showed two melt peaks only at low template input, what would you suspect?