Primer dimer test
Primer-dimers can bias qPCR results, so a test for primer-dimers should be performed during assay development to ensure that only the target sequence is amplified and quantified.
The effect of primer dimers
Checking for primer dimers is crucial when developing a qPCR assay. Primer dimers form when two primers bind to each other instead of the target DNA. This leads to the polymerase amplifying the primers themselves, creating short, nonspecific DNA fragments. This phenomenon reduces reaction efficiency and makes the assay less accurate.
Primer dimers can significantly bias the results in SYBR Green-based assays (or assays using other DNA-binding dyes). Since these dyes intercalate with double-stranded DNA (dsDNA) and emit fluorescence, primer dimers, which are also dsDNA, bind to the dye, producing a fluorescence signal. The signal from primer dimers results in false positives, as the amplified primer dimers are indistinguishable from the target amplicon in the fluorescence readout.
In probe-based assays, primer dimers have a different impact. These assays rely on the cleavage of a specific probe to generate a fluorescence signal rather than the presence of dsDNA. However, primer-dimer formation can still interfere indirectly by consuming reaction components such as dNTPs, primers, and polymerase. Primer amplification reduces the efficiency of specific target amplification and can lead to biased results.
Primer dimers can also limit the assay’s ability to detect low-abundance targets, which is critical for applications such as:
- Gene therapy biodistribution, shedding, and pharmacokinetics.
- Detection of circulating tumor DNA (ctDNA).
- Monitoring minimal residual disease (MRD) in cancer.
Testing for and minimizing primer dimers is essential during assay development to ensure reliable and accurate qPCR results.
How to check for primer dimers
The standard method to check for primer dimers in SYBR Green-based qPCR is to perform a melting curve analysis. This post-amplification analysis provides the amplified product’s melting temperature (Tm). A single, sharp peak in the melting curve indicates that a specific product has been amplified, while multiple peaks suggest the presence of nonspecific amplicons or primer dimers.
Gel electrophoresis is performed to confirm the presence of primer dimers in probe-based assays. This technique allows you to identify short, primer-dimer-sized bands after amplification.
A faster and more time-saving alternative is to include BOXTO in the reaction. BOXTO can detect nonspecific amplification or primer dimers directly during the qPCR run, providing immediate feedback on assay specificity.
A fast and easy primer dimer test
BOXTO is a fluorescent dye that binds to double-stranded DNA (dsDNA) and emits fluorescence in the JOE channel. It can be used alongside fluorescent probes without interfering with the specific probe signal, eliminating the need for post-amplification gel electrophoresis.
BOXTO enables real-time tracking of overall DNA amplification, including target-specific and nonspecific products, such as primer dimers. It is particularly useful for confirming that target-specific amplification is the only amplification occurring, even when the probe signal is absent. BOXTO provides a quick and efficient way to ensure the specificity and reliability of your qPCR assay necessary for low-abundance targets.
The best results require the best reagents.
At TATAA Biocenter, we specialize in designing, optimizing, and validating qPCR and dPCR assays for cell and gene therapy bioanalysis. Whether the goal is to measure how a gene product distributes in the body, assess environmental shedding, or evaluate transgene expression across different tissues, the assay must perform with precision and reliability.
As the authors of the MIQE guidelines, TATAA Biocenter has always prioritized developing and using control assays and reagents that ensure the highest quality and accuracy in every result.
