Methods for detecting nucleic acid sequences using evanescent wa

Details Methods for detecting nucleic acid sequences using evanescent wa Methods for detecting nucleic acid sequences using evanescent wa

1994-02-23

1998-05-12

Marschel, Ardin H.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 435810, 436501, 422 50, 422 681, 935 77, 935 78, C12Q 168

Patent

active

057503370

DESCRIPTION:

BRIEF SUMMARY
This is a 371 filing of PCT/GB92/01698, filed Sep. 16, 1992.
The present invention relates a method for the detection, identification and/or quantification of plant or animal tissues, microorganisms or cell free RNA or DNA and to reagents and detector apparatus adapted for performing said method. The method particularly uses Total Internal Reflection Fluoresence (TIRF) to measure hybridization of analyte RNA or DNA with RNA or DNA that is associated with an evanescent wave detector waveguide.
Gene probe assays, using nucleic acid hybridization, are an alternative to immunoassays in the detection and identification of biological materials. The specificity of gene probes for their targets can be controlled much more easily than is possible using protein-based binding phenomena and, when coupled with the polymerase chain reaction to pre-amplify the target material, extreme sensitivity can be obtained. Current techniques using gene probes are slow, taking from hours to days to produce a result. Biosensors offer an alternative route to fast gene probe assays, but the only reports so far on gene probe biosensor assays are those using surface plasmon resonance (Evans & Charles (1990); Abstracts of 1st World Congress on DNA probes and immunoassay; Pollard-Knight et al (1990) Ann. Biol. Clin, 48 642-646).
Evanescent wave biosensors, which use the phenomenon of TIRF for detection (Sutherland & Dahne, (1987) J. Immunol. Meth., 74, 253-265), have previously been used with proteins as the biological recognition element. Antibodies have been used to detect the binding of fluorescent-labelled antigen (Eldefrawi et al (1991) Biosensors & Bioelectronics, 6, 507-516) using acetylcholine receptors to study the binding of acetylcholine and cholinesterase inhibitors. Other groups (Poglitsch & Thompson (1990) Biochemistry, 29, 248-254) have measured the binding of antibody to F.sub.c epitopes.
The present invention provides a method for carrying out gene probe assays with an evanescent wave biosensor and provides a TIRF waveguide which is adapted for carrying out said method when incorporated within an evanescent wave biosensor device.
Evanescent wave detectors exploit the TIRF phenomenon to provide a sensitive method for detecting reactions at the surface of waveguides. The waveguide may take various forms but typically will be a prism, slab or fibre. The reaction to be used to measure the target molecule may be monitored, for example, through measuring the fluorescence changes on binding or desorption of fluorescent species or by the generation of fluorescent species by enzymic or chemical means. Several patents have been published that cover the use of evanescent wave detectors with immunoassay systems as the biological sensing element (eg. U.S. Pat. No. 4,582,809) but the inherent limitations in the immuno-reagents have not allowed the full capabilities of the sensor to be exploited.
The present invention provides a method for the detection, identification and/or quantification of a material selected fom plant or animal tissue, microorganisms or cell free RNA or DNA comprising: oligonucleotide sequence characteristic of the RNA or DNA of the material, immobilised on the surface of an evanescent wave detector apparatus waveguide; DNA or RNA material derived therefrom, under conditions whereby DNA or RNA having the characteristic oligonucleotide sequence will hybridize with it; nucleic acid material derived from the sample with a fluorescently detectable agent, before, during or on completion of any hybridization with DNA or RNA as provided in step (ii), such that the fluorescently detectable agent becomes bound to the hybridized product but not to unhybridized immobilized oligonucleotide; the evanescent wave detector apparatus and relating the amount of that to the presence, identity and/or amount of the material.
In one preferred form of the present invention the fluorescently detectable agent is a fluorescently detectable intercalating dye capable of being incorporated into the duplex of the hybridization product. In this

REFERENCES:
patent: H1398 (1995-01-01), Campbell
patent: 4582809 (1986-04-01), Block et al.
patent: 4683195 (1987-07-01), Mullis et al.
patent: 5242797 (1993-09-01), Hirschfield
Graham et al Biosensors & Bioelectronics, 7 (1992) 487-493) Gene probe assays on a fibre-optic evanescent wave biosensor.

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