Real-time Detection of Nucleic Acid Interactions by Total Internal Reflection Fluorescence

Anal Chem. 2003 May 15;75(10):2414-20. doi: 10.1021/ac0206519.

Abstract

This paper describes the development of an optical readout system for the real-time analysis of fluorescent-labeled DNA microarrays is described. The system is targeted toward research applications in genomics, agriculture, and life sciences, where the end-point detection of state-of-the-art readout systems does not provide sufficient information on the hybridization process. The hybridization progress of molecules from the liquid phase in a flow cell to immobilized oligonucleotides on a transducer surface can be observed. The excitation of fluorochromes is realized by a semiconductor laser, and the fluorescence emission is collected by a cooled CCD camera. Quantitative data can be extracted from the images for analysis of the microarray. For the signal transduction, the principle of total internal reflection is used. With a multiple internal reflection arrangement, the sensor chip was adapted to the standard microscope slide format and a homogeneous evanescent illumination of the active area of the sensor surface was achieved. An application measurement was carried out with this readout system. The hybridization of Cy5-labeled 30-mer single-stranded oligonucleotides to fully complementary immobilized strands was observed in real time. A kinetic analysis was demonstrated with the recorded data. Melting curves of a 140-mer PCR product from a hemochromatosis patient sample hybridized to immobilized wild-type mutant 15- and 17-mer oligonucleotides were recorded and single-point mutations could be detected.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Hemochromatosis / genetics
  • Humans
  • Kinetics
  • Nucleic Acid Hybridization
  • Nucleic Acids / analysis
  • Nucleic Acids / genetics
  • Nucleic Acids / metabolism*
  • Oligonucleotide Array Sequence Analysis / methods
  • Oligonucleotides / analysis
  • Oligonucleotides / chemistry
  • Optics and Photonics
  • Point Mutation
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods*

Substances

  • Nucleic Acids
  • Oligonucleotides