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

Autor:	Ángel Maquieira, Luis Antonio Tortajada-Genaro, Ana Lázaro
Rok vydání:	2021
Předmět:	Mutant 02 engineering and technology Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Polymorphism Single Nucleotide 01 natural sciences Biochemistry Cell Line Analytical Chemistry Allele-selective qPCR Bioanalytical methods DNA variant detection KRAS oncogene Mutation genotyping chemistry.chemical_compound Limit of Detection QUIMICA ANALITICA Genetic variation medicine Humans Point Mutation Allele KRAS oncogene DNA variant detection Oncogene Oligonucleotide Point mutation 010401 analytical chemistry Reproducibility of Results 021001 nanoscience & nanotechnology Molecular biology 0104 chemical sciences Genes ras chemistry Bioanalytical methods Allele-selective qPCR KRAS Colorectal Neoplasms 0210 nano-technology Mutation genotyping DNA
Zdroj:	RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia instname ANALYTICAL AND BIOANALYTICAL CHEMISTRY r-IIS La Fe. Repositorio Institucional de Producción Científica del Instituto de Investigación Sanitaria La Fe
ISSN:	1618-2650 1618-2642
DOI:	10.1007/s00216-021-03229-3
Popis:	[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. 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)
Databáze:	OpenAIRE
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