Solid-Phase Hybridization Assay for Detection of Mutated Cancer DNA by Fluorescence

Methods Mol Biol. 2020:2063:37-44. doi: 10.1007/978-1-0716-0138-9_4.

Abstract

We report a straightforward protocol for the detection of mutated DNA extracted from cancer cells. The assay combines a step-wise solid-phase hybridization and a readout by fluorescence emission. We detect a single-nucleotide polymorphism in two human oncogenes, BRAF and EGFR, and reach a limit of the detection of 300 pM by conventional fluorometry. The protocol described herein may be used as a foundation for development of automatic optimized assays capable for detection of mutant DNA and RNA in vitro and in cells.

Keywords: DNA oncogene; Fluorescent oligonucleotides; Fluorometry; Hybridization assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / genetics*
  • ErbB Receptors / genetics
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Fluorometry / methods
  • Humans
  • Limit of Detection
  • Neoplasms / genetics*
  • Nucleic Acid Hybridization / methods
  • Perylene / chemistry
  • Polymorphism, Single Nucleotide / genetics*
  • Proto-Oncogene Proteins B-raf / genetics*

Substances

  • Fluorescent Dyes
  • Perylene
  • DNA
  • EGFR protein, human
  • ErbB Receptors
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf