COMplementary Primer ASymmetric PCR (COMPASTM-PCR), a counter intuitive primer design for PCR

Sammendrag

Since it was developed in the mid 80’, the Polymerase Chain Reaction (PCR) has established itself as a central tool for molecular biology. Instrumentation, reagents as well as methodology have regularly improved and expanded the capabilities of this technology. Limitations inherent to DNA and kit chemistries have generated guidelines for PCR-assay development. For instance, non-target amplification and in particular primer complementarity leading to Primer Dimer formation is a well-known limiting factor to be reckoned with. Although various strategies have helped improved general robustness of PCR assays, primer complementarity usually is carefully avoided when designing PCR by using ad hoc software. Indeed, 3'-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling any possible further involvement in traditional target amplification. However, a 5'-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also competing with target amplification. In this work we show that 5'-complementarity between primers may be exploited in a new asymmetric PCR method, the COMplementary-Primer-Asymmetric (COMPASTM)-PCR, to achieve effective double strand target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. Using asymmetric primer concentration is not a new approach but has, to our knowledge, only been previously used for enhancing the production of single strand amplicon from a target sequence, i.e. for probe detection assays. In the presence of the target sequence, COMPAS-PCR initiates target linear amplification with the excess primer, hence progressively changing the stoichiometry of the reaction so that priming of the limiting primer to the target strand and target extension of the complementary strand is gradually favored. This general principle was developed using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. This initial application, published in PLoS One (doi:10.1371/journal.pone.0165468), was designed so that both primers are complementary to the same genomic sequence target. This approach may in principle be applied to any tandem direct repeats DNA motifs of interest as target sequences. Ribosomal genes and in particular the 5S rDNA tandemdirect repeats are found in all eukaryotic cells and are therefore suitable for developing new specific complementary-primer assays. We believe that further understanding and modelling of these COMPAS-PCR principles could be incorporated in primer design software. This small paradigm shift, using highly complementary primers for PCR, should help develop or improve PCR assays, increasing design possibilities available to the molecular scientist.