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. 2021 Feb 18;4:227. doi: 10.1038/s42003-021-01751-9

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Heterogeneous genomes contain single-nucleotide variants within the probe-binding site that prevent the use of probe-based qPCR for DRM discrimination. b PANDAA primers overlap with the probe-binding site, adapting the nucleotides proximal to the DRM of interest that would otherwise abrogate probe hybridization. c, d As qPCR proceeds, the newly generated amplicon will contain probe-binding sites flanking the DRM that are perfectly complementary to the probe. e PANDAA primers contain two key features: a 3′ ADR that is matched to the probe-binding site and a pan-degenerate (PDR) region that incorporates the nucleotide degeneracy observed in the primer-binding site of the target. The PDR accounts for the high degree of variability in primer-binding sites. LNA bases at 100% conserved positions in the PDR offset the thermodynamic instability of mismatches between the primer ADR and the template.