Mispriming

Mispriming refers to the initiation of DNA synthesis by a primer at an unintended site, producing off-target products in PCR, sequencing, or cDNA synthesis. It occurs when primers bind partially or completely to non-target sequences, or when reaction conditions promote non-specific annealing, primer-dimer formation, or template switching.

In PCR, mispriming can yield non-specific amplification bands, reduced yield of the target product, and artifacts such as chimeric sequences. It is influenced by sequence similarity between the primer and unintended templates, repetitive or low-complexity regions, suboptimal annealing temperatures, primer length and design flaws, and contamination.

Detection of mispriming typically relies on analytical methods that reveal off-target products. Gel electrophoresis may show unexpected bands; sequencing of amplicons can reveal non-target sequences; melt-curve analysis or probe-based assays in qPCR can indicate multiple products or non-specific amplification; in sequencing libraries, mispriming can show up as unexpected reads or chimeras.

Prevention and mitigation focus on primer design and reaction conditions. Use in silico checks to ensure primer specificity against the target genome or transcriptome, avoiding regions with homology to non-target sequences. Design primers of suitable length (roughly 18–25 nucleotides), balanced GC content (about 40–60%), and minimal propensity for hairpins or dimers. Optimize annealing temperature and Mg2+ concentration; use hot-start polymerase; consider touchdown or nested PCR to improve specificity. For cDNA synthesis, select appropriate priming strategies (gene-specific or anchored oligo(dT)) to reduce unintended priming. Proper controls are essential to distinguish true target amplification from misprimed artifacts.