Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene

Autores de IIS La Fe

• Sarai Palanca Suela

  Autor

• Ángel Maquieira Catalá

  Autor

• Luis A. Tortajada Genaro

  Autor

Participantes ajenos a IIS La Fe

• Martorell, S

Grupos

• Investigación Clínica y Traslacional en Cáncer

  

  Leader

  José Gómez Codina

• Unidad Mixta de Investigación en Nanomedicina y Sensores

  Leader

  Ramón Martínez Máñez

Abstract

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 degrees C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.

Keywords

• Recombinase polymerase amplification; Blocking agent; Mutations in PIK3CA oncogene; Gel electrophoresis; Colorimetric array