Real-time detection of nucleic acid interactions by total internal reflection fluorescence.
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.
Lehr
H P
HP
Fraunhofer-Institute of Physical Measurement Techniques IPM, Heidenhofstrasse 8, D-79110 Freiburg, Germany. HP.Lehr@t-online.de
Reimann
M
M
Brandenburg
A
A
Sulz
G
G
Klapproth
H
H
D006432:Hemochromatosis / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D055095:Optics and Photonics; D017354:Point Mutation; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D009696:Nucleic Acids; D009841:Oligonucleotides