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PLoS One. 2016 Oct 26;11(10):e0165468. doi: 10.1371/journal.pone.0165468. eCollection 2016.

COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids.

Author information

1
Department of Aquaculture, Norwegian Institute for Water Research, Oslo, 0349, Norway.

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.

PMID:
27783658
PMCID:
PMC5082663
DOI:
10.1371/journal.pone.0165468
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

I have read the journal's policy and the author of this manuscript has the following competing interest: part of this work is used in patent application WO 2014/168484 A1 entitled “COMPAS-PCR method and methods for detecting, identifying or monitoring salmonid species”. My employer, the Norwegian Institute for Water Research, is the sole applicant for this patent. This does not alter my adherence to PLOS ONE policies on sharing data and materials.

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