Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene


An accurate genetic diagnostic is key for adequate patient management and the suitability of healthcare systems. The scientific challenge lies in developing methods to discriminate those patients with certain genetic variations present in tumor cells at low concentrations. We report a method called enhanced asymmetric blocked qPCR (EAB-qPCR) that promotes the blocker annealing against the primer-template hybrid controlling thermal cycling and reaction conditions with nonmodified oligonucleotides. Real-time fluorescent amplification curves of wild-type alleles were delayed by about eight cycles for EAB-qPCR, compared to conventional blocked qPCR approaches. This method reduced the amplification of native DNA variants (blocking percentage 99.7%) and enabled the effective enrichment of low-level DNA mutations. Excellent performance was estimated for the detection of mutated alleles in sensitivity (up to 0.5% mutant/total DNA) and reproducibility terms, with a relative standard deviation below 2.8%. The method was successfully applied to the mutational analysis of metastatic colorectal carcinoma from biopsied tissues. The determined single-nucleotide mutations in the KRAS oncogene (codon 12–13) totally agreed with those obtained from next-generation sequencing. EAB-qPCR is an accurate cheap method and can be easily incorporated into daily routine to detect mutant alleles. Hence, these features are especially interesting to facilitate the diagnosis and prognosis of several clinical diseases.

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The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-FPI-2017 PhD grant), the Spanish Ministry of Economy and Competitiveness (MINECO project CTQ2016-75749-R), and European Regional Development Fund (ERDF).

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Correspondence to Luis A. Tortajada-Genaro.

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Research involving human subjects complied with all relevant national regulations and institution policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the authors’ institutional review board. Informed consent was obtained from all individuals included in this study.

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Lázaro, A., Tortajada-Genaro, L.A. & Maquieira, Á. Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene. Anal Bioanal Chem 413, 2961–2969 (2021). https://doi.org/10.1007/s00216-021-03229-3

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  • Bioanalytical methods
  • Allele-selective qPCR
  • KRAS oncogene
  • Mutation genotyping
  • DNA variant detection