4.9.1. Primer-dimers and other PCR artefacts

PCR is susceptible to qualitative and quantitative errors caused by the accidental, and highly efficient, amplification of short non-target PCR templates, especially when there is little target template available. Such artefactual amplifications arise from fleeting, partial complementarity of the primers with non-target templates, or among the primers themselves (SantaLucia, 2007). The latter version is called ‘primer-dimer’ and is formed through (self)-complementarity at the 3’ end of the amplification primers. For example, if one primer ends in N16AC-3’ and another primer in N16GT-3’, the two primers can form a short template through the pairing of these two 3’ base-pairs (Fig. 1A). If a primer ends in complementary bases (N16GC-3’ or N16AT-3’) it could even create a 2-bp overlap with itself (Fig. 1B), generating a short amplifiable fragment. The risk of primer-dimer increases with the number of unique primers in a reaction, such as in multiplex PCR (see section 4.7.5; “Multiplex RT-(q)PCR”). Primer-dimer is identified if a product is produced in a template-free reaction. If PCR artefacts are only produced in samples, but not template-free controls, then the cause is less clear, involving most likely other nucleic acids molecules present in the samples. In both cases, the easiest solution is to design new primers and test these experimentally (SantaLucia, 2007).

Fig. 1. Formation of primer-dimer through complementarity between the 3’ ends of two primers (5A) or self-complementarity of the 3’ end of a single primer (5B).

Figure 1