DNA probe diffraction assay and reagents

• US 5,089,387 A
• Filed: 07/07/1988
• Issued: 02/18/1992
• Est. Priority Date: 07/07/1988
• Status: Expired due to Fees

• Current Assignee: Cytyc Prenatal Products Corp. (Hologic Incorporated)
• Original Assignee: Cytyc Prenatal Products Corp. (Hologic Incorporated)
• Inventors: Calenoff, Emanuel, Lee, John, Trebino, Rick, Gustafson, Eric K., Tsay, Yuh-Geng
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): STUCKER, JEFFREY J
• Application Number:
  US07/216,691
• Time in Patent Office: 1,321 Days
• Field of Search: 422/55, 436/518, 436/524, 436/531, 435/6, 424/250
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 2021/757 using immobilised reagents
• CPC Class Codes: G01N 21/4788 Diffraction for sizing particles G01N15/0205
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

The assay of the subject invention uses DNA sequences as probes in a nucleic acid hybridization diffraction assay, to detect specific DNA sequences in a sample. Diffraction assay methodologies are applied to determine the presence and amount of analyte.

This invention involves a discovery in the areas of supporting surfaces for a biogrid or biograting which provide greatly reduced non-specific hybridization and binding. A preferred process of this invention involves manufacturing a biograting for use in a light diffraction assay, and comprises adhering a uniform layer of hybridizing reagent comprising a nucleotide sequence on a smooth, solid surface and exposing the surface to UV radiation through a shadow mask with a diffraction grating pattern of lines to selectively deactivate the hybridizing reagent, leaving a biological diffraction grating design of lines of active hybridizing reagent. The smooth, solid surface is preferably selected from the group consisting of polysilicon and single crystalline silicon surfaces.

The diffraction hybridizing assay method of this invention for determining the presence or quantity of an analyte in an aqueous sample comprises contacting a nucleic acid sequence diffraction biogrid with the sample under proper circumstances and for a sufficient time to permit nucleic acid hybridization between a nucleic acid sequence probe and an analyte; separating the biogrid from the sample; illuminating the biogrid with light from a light source; and determining the light diffracted by the diffraction hybridization assay surface.

Claims

1. A biograting for use in a light diffraction assay comprising a smooth surface having on said surface, a biological diffraction grating design of lines of active hybridizing reagent.

2. The biograting of claim 1 wherein the hybridizing reagent is a nucleotide sequence.

3. The biograting of claim 1 wherein the smooth surface is selected from the group consisting of single crystalline silicon, aluminum, epitaxial silicon coatings, silicon nitride coatings, silicon dioxide coatings, and polysilicon coatings.

4. The biograting of claim 3 wherein the smooth surface is selected from the group consisting of polysilicon and single crystalline silicon surfaces.

5. The biograting of claim 1 wherein the biological diffraction grating design of lines of active hybridizing reagent is substantially non-light disturbing.

6. The biograting of claim 1 wherein the biological diffraction grating design of lines becomes light-disturbing in the presence of hybridization with analyte.

7. A process for manufacturing the biograting of claim 1, comprising adhering a uniform layer of hybridizing reagent on a smooth, solid surface and exposing the surface to UV radiation through a shadow mask with diffraction grating lines to selectively deactivate the hybridizing reagent leaving a biological diffraction grating design of lines of active hybridizing reagent.

8. A process for manufacturing a biograting for use in a light diffraction assay, comprising:

(a) adhering a uniform layer of hybridizing reagent on a smooth, solid surface; and

(b) exposing the surface to UV radiation through a shadow mask with diffraction grating lines to selectively deactivate the hybridizing reagent to leave a biological diffraction grating design of lines of active hybridizing reagent.

9. The biograting of claim 8 wherein the hybridizing reagent is a nucleotide sequence.

10. The biograting of claim 8 wherein the smooth surface is selected from the group consisting of single crystalline silicon, aluminum, epitaxial silicon coatings, silicon nitride coatings, silicon dioxide coatings, and polysilicon coatings.

11. The biograting of claim 10 wherein the smooth surface is selected from the group consisting of polysilicon and single crystalline silicon surfaces.

12. The biograting of claim 8 wherein the biological diffraction grating design of lines of active hybridizing reagent is substantially non-light disturbing.

13. The biograting of claim 8 wherein the biological diffraction grating design of lines becomes light-disturbing in the presence of hybridization with analyte.

14. A process for manufacturing the biograting of claim 8, comprising adhering a uniform layer of hybridizing reagent on a smooth, solid surface and exposing the surface to UV radiation through a shadow mask with diffraction grating lines to selectively deactivate the hybridizing reagent leaving a biological diffraction grating design of lines of active hybridizing reagent.

15. An assay method for determining the presence or quantity of an analyte nucleotide sequence in an aqueous sample comprising

(a) contacting an assay surface with the sample for a time sufficient to permit binding of hybridizing reagent and analyte, the assay surface having on said surface, a light disturbing design of substantially non-light disturbing hybridizing reagent thereon, the hybridizing reagent being selected to hybridize selectively with the analyte;

and

(b) illuminating the assay surface with light from a light source, and determining the light diffracted by the assay surface.

16. The assay method of claim 15 wherein the analyte nucleotide sequence is derived from a body tissue.

17. The assay method of claim 15 wherein the analyte nucleotide sequence is present in a body fluid.

18. The assay method of claim 17 wherein the analyte nucleotide sequence is present in urine, blood serum, blood plasma, milk, cerebrospihal fluid, mucus or sputum.

19. The assay method of claim 17 wherein the analyte nucleotide sequence is present in bacteria, viruses, fungi, algae, animal or plant cells.

20. The assay method of claim 15 wherein the analyte is a nucleotide sequence having origin in a fetal cell, adult cell, bacteria, virus, Mycoplasmatales, spore, algae, parasite, or yeast.