title: COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids.
abstract: Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3'-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5'-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5'-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated 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. Specificity was enhanced by using 3'-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered.
authors: Department of Aquaculture, Norwegian Institute for Water Research, Oslo, 0349, Norway.
authors: Marc B
authors: http://orcid.org/0000-0001-6385-663X
authors: MB
authors: Anglès d'Auriac
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D020317:Salmo salar / Q000235:genetics; D012474:Salmon / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012341:RNA, Ribosomal, 5S