PrimerDimer

Primer-dimer is a common PCR artifact in which primers anneal to each other rather than to the target template, and are extended by the DNA polymerase to form short, non-target amplification products. It can occur in standard PCR, RT-PCR, and quantitative PCR (qPCR) assays and can interfere with amplification efficiency and detection.

Formation of primer-dimers arises when primers contain complementary regions, especially at their 3' ends, allowing them to anneal to each other or to form self-dimers. If the 3' ends are aligned in a way that permits extension, the polymerase can copy the primer-primer complex, generating short double-stranded fragments typically tens of base pairs long. These products compete with the desired template for reagents and can accumulate during cycling.

The presence of primer-dimers can reduce yield of the intended product and, in qPCR, may contribute to non-specific fluorescence signals that skew Ct values and complicate data interpretation. In conventional PCR, they often appear as faint, small bands on a gel or appear in the reaction as a smear near the bottom of the gel.

Prevention and mitigation strategies focus on primer design and reaction optimization. Avoid complementary sequences between primer pairs, especially at 3' ends; design primers with balanced Tm and minimal potential for hairpins or dimers. Use primer design software, validate with melt-curve analysis, and employ hot-start polymerases or higher annealing temperatures. Adjust primer concentrations, Mg2+ levels, or switch to touchdown PCR if necessary. In some cases, redesigning primers is the most reliable solution to eliminate primer-dimers.