Nucleotide-selective amplification and array-based detection for identifying multiple somatic mutations.

Autores de IIS La Fe

• Luis A. Tortajada Genaro

  Autor

• Ángel Maquieira Catalá

  Autor

Participantes ajenos a IIS La Fe

• Lazaro, Ana
• Martorell, Sara

Grupos

• Unidad Mixta de Investigación en Nanomedicina y Sensores

  Leader

  Ramón Martínez Máñez

Abstract

In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are responsive to therapies. As an alternative to one-by-one detection or massive sequencing, the presented genotyping tool determines multiple polymorphic sequences that vary a single nucleotide. The biosensing method includes an effective enrichment of mutant variants and selective recognition by colorimetric DNA arrays. The proposed approach is the hybridization between sequence-tailored probes and products from PCR with SuperSelective primers to discriminate specific variants in a single locus. A fluorescence scanner, a documental scanner, or a smartphone captured the chip images to obtain spot intensities. Hence, specific recognition patterns identified any single-nucleotide change in the wild-type sequence overcoming qPCR methods and other array-based approaches. Studied mutational analyses applied to human cell lines provided high discrimination factors, the precision was 95%, and the sensitivity was 1% mutant of total DNA. Also, the methods showed a selective genotyping of the KRAS gene from tumorous samples (tissue and liquid biopsy), corroborating results by NGS. The developed technology supported on low-cost robust chips and optical reading provides an attractive pathway toward implementing fast, cheap, reproducible discrimination of oncological patients.

Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

Keywords

• Allele-specific techniques; Array sensing; DNA chip technology; Mutations in oncogenes