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

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

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Nombre: Lazaro-ZaragozaTo ...
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dc.contributor.author Lázaro-Zaragozá, Ana es_ES
dc.contributor.author Tortajada-Genaro, Luis Antonio es_ES
dc.contributor.author Maquieira Catala, Ángel es_ES
dc.date.accessioned 2021-12-30T19:27:08Z
dc.date.available 2021-12-30T19:27:08Z
dc.date.issued 2021-05 es_ES
dc.identifier.issn 1618-2642 es_ES
dc.identifier.uri http://hdl.handle.net/10251/179192
dc.description.abstract [EN] 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. es_ES
dc.description.sponsorship 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) es_ES
dc.language Inglés es_ES
dc.publisher Springer-Verlag es_ES
dc.relation.ispartof Analytical and Bioanalytical Chemistry es_ES
dc.rights Reserva de todos los derechos es_ES
dc.subject Bioanalytical methods es_ES
dc.subject Allele-selective qPCR es_ES
dc.subject KRAS oncogene es_ES
dc.subject Mutation genotyping es_ES
dc.subject DNA variant detection es_ES
dc.subject.classification QUIMICA ANALITICA es_ES
dc.title Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene es_ES
dc.type Artículo es_ES
dc.identifier.doi 10.1007/s00216-021-03229-3 es_ES
dc.relation.projectID info:eu-repo/grantAgreement/GVA//GVA-FPI-2017/ es_ES
dc.relation.projectID info:eu-repo/grantAgreement/AEI//CTQ2016-75749-R//BIOSENSORES HOLOGRAFICOS. PRUEBA DE CONCEPTO Y DEMOSTRACION EN APLICACIONES CLINICAS/ es_ES
dc.rights.accessRights Abierto es_ES
dc.contributor.affiliation Universitat Politècnica de València. Departamento de Química - Departament de Química es_ES
dc.description.bibliographicCitation Lázaro-Zaragozá, A.; Tortajada-Genaro, LA.; Maquieira Catala, Á. (2021). Enhanced asymmetric blocked qPCR method for affordable detection of point mutations in KRAS oncogene. Analytical and Bioanalytical Chemistry. 413(11):2961-2969. https://doi.org/10.1007/s00216-021-03229-3 es_ES
dc.description.accrualMethod S es_ES
dc.relation.publisherversion https://doi.org/10.1007/s00216-021-03229-3 es_ES
dc.description.upvformatpinicio 2961 es_ES
dc.description.upvformatpfin 2969 es_ES
dc.type.version info:eu-repo/semantics/publishedVersion es_ES
dc.description.volume 413 es_ES
dc.description.issue 11 es_ES
dc.identifier.pmid 33619642 es_ES
dc.relation.pasarela S\429717 es_ES
dc.contributor.funder Generalitat Valenciana es_ES
dc.contributor.funder AGENCIA ESTATAL DE INVESTIGACION es_ES
dc.contributor.funder European Regional Development Fund es_ES
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