Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers

Lab Chip. 2013 Jun 7;13(11):2106-14. doi: 10.1039/c3lc50129a.

Abstract

Here, we first present an isothermal solid-phase amplification/detection (ISAD) technique for the detection of single-point mutations that can be performed without labelling in real-time by utilizing both silicon microring-based solid-phase amplification and isothermal recombinase polymerase amplification (RPA). The ISAD technique was performed on a silicon microring device with a plastic chamber containing 10 μL of the reaction mixture, and characterized with an assay for the detection of the HRAS (Harvey RAS) gene single-point mutation. For the solid-phase amplification, the primer of the gene was directly attached to the surface of the device via an amine modification reaction. The amplified DNA was detected, without a label, by measuring the optical wavelength shift of the silicon microring resonator during the reaction. We demonstrated that the sensitivity of the ISAD technique was 100-times higher than that of RPA and conventional PCR methods. Moreover, this technique can be used to distinguish a single-point mutation of the HRAS gene via target amplification. This novel DNA amplification/detection technique will be useful for the detection of sequence alterations such as mutations and single-nucleotide polymorphisms as DNA biomarkers in human diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Neoplasm / genetics
  • Humans
  • Neoplasms / genetics*
  • Nucleic Acid Amplification Techniques / instrumentation*
  • Point Mutation
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • Recombinases / metabolism
  • Silicon / chemistry
  • Temperature*
  • Time Factors

Substances

  • DNA, Neoplasm
  • Recombinases
  • Proto-Oncogene Proteins p21(ras)
  • Silicon