Method and structures employing chemically-labelled polynucleotide probes

• US 4,994,373 A
• Filed: 07/20/1989
• Issued: 02/19/1991
• Est. Priority Date: 01/27/1983
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Johnston, Kenneth H., Thalenfeld, Barbara E., Stavrianopoulos, Jannis G., Kirtikar, Dollie
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/385,986
• Time in Patent Office: 579 Days
• Field of Search: 536/27, 436/800, 436/810, 935/77, 935/78, 935/86, 935/87
• US Class Current: 435/6.16
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Polynucleotide sequences in a sample of biological or nonbiological material are detected by a method involving fixing of the sequences on a solid support and forming an entity between the fixed sequences and chemically-labeled polynucleotide or oligonucleotide probes having a sequence complementary to the fixed sequence for determining the identification and/or presence of the target polynucleotide sequences. The chemical label covalently or noncovalently attached to the probe comprises a signalling moiety capable of generating a soluble signal detectable by spectrophotometric assay techniques.

Claims

1. A method for detecting a polynucleotide sequence which comprises:

fixing said polynucleotide sequence to a solid support which comprises or is contained within a transparent or translucent, non-porous system, such that a single-strand of the polynucleotide is capable of hybridizing to complementary nucleic acid sequences;

forming an entity comprising said polynucleotide sequence hybridized to a polynucleotide or oligonucleotide probe, said probe having attached thereto a chemical label further comprising a signalling moiety capable of generating a soluble signal; and

generating and detecting said soluble signal.

2. The method according to claim 1, wherein said detecting step comprises spectrophotometric techniques.

3. The method according to claim 1, wherein said soluble signal is selected from the group consisting of a colored product, a chemiluminescent product and a fluorescent product.

4. The method according to claim 1, wherein said signalling moiety is selected from the group consisting of an enzyme, a chelating agent and a co-enzyme.

5. The method according to claim 1, wherein said solid support is selected from the group consisting of glass, plastic, polystyrene, polyethylene, dextran and polypropylene.

6. The method according to claim 1, wherein said polynucleotide sequence is directly fixed to said solid support.

7. The method according to claim 6, wherein said polynucleotide sequence is fixed to said solid support in single stranded form.

8. The method according to claim 1, wherein said signalling moiety is attached to said polynucleotide or oligonucleotide probe through the formation of a complex.

9. The method according to claim 8, wherein said complex is selected from the group consisting of biotin and avidin, biotin and streptavidin, and a sugar and a lectin.

10. The method according to claim 1, wherein said forming step further comprises washing to remove said polynucleotide or oligonucleotide probes that do not form said entity.

11. The method according to claim 10, wherein said forming step further comprises attaching said signalling moiety to said polynucleotide or oligonucleotide probe after said washing step.

12. The method according to claim 11, which further comprises separating free signalling moieties from said attached signalling moieties.

13. The method according to claim 1, wherein said detecting step further comprises generating said soluble signal in a device capable of transmitting light therethrough for the detection of said soluble signal by spectrophotometric techniques.

14. The method according to claim 13, wherein said device is selected from the group consisting of a well, a tube, a cuvette and an apparatus which comprises a plurality of said wells, tubes or cuvettes.

15. The method according to claim 13, wherein said soluble signal is selected from the group consisting of a colored product, a chemiluminescent product, and a fluorescent product.

16. The method according to claim 13, wherein said solid support and said device are composed of the same materials.

17. A device for detecting a polynucleotide sequence according to the method of claim 1, which device comprises a solid support, having said polynucleotide sequence fixed thereto in hybridizable form.

18. A kit for detecting a polynucleotide sequence, which comprises the device of claim 17 in packaged combination with a container of an oligonucleotide or polynucleotide probe, having covalently attached thereto a chemical label comprising a signalling moiety capable of generating a soluble signal.

19. The kit of claim 18, wherein said soluble signal is a colored product or a fluorescent product.

20. The method according to claim 1 wherein part of the solid support is modified to facilitate fixing of the polynucleotide sequence to the solid support by the sequential steps of:

(a) heating or boiling the solid support in dilute nitric acid for about 45 minutes;

(b) washing or rinsing the solid support with distilled water;

(c) drying the solid support at about 115°

C., for about 24 hours;

(d) incubating the solid support in contact with 10% gamma-aminopropyltriethoxysilane for about two to three hours at about 45°

C.;

(e) washing with water; and

(f) drying overnight at a temperature of about 100°

C.

21. The method according to claim 1 wherein part of the solid support is modified to facilitate fixation of the polynucleotide sequence to the solid support by treating the solid support with a coating of an epoxy resin.

22. The method according to claim 21 wherein part of the solid support is modified to facilitate fixation of the polynucleotide sequence to the solid support by treating the support with the epoxy resin by the following sequential steps;

(a) applying an epoxy glue in solution with ethanol to the solid support; and

,

(b) evaporating the ethanol by heating to a temperature of about 37°

C. to provide a polyamine polymeric coating on the solid support.

23. The method according to claim 1 wherein said polynucleotide sequence is fixed to said solid support in double-stranded form, and denatured into single-stranded form prior to the hybridization step.

24. The method according to claim 1 wherein said polynucleotide sequence in double-stranded form is denatured, and fixed to said solid support in single-stranded form prior to the hybridization step.

25. The method according to claim 1 wherein said polynucleotide sequence to be detected is in single-stranded form and is indirectly bound to said solid support by sandwich hybridization.

26. The method according to claim 1 wherein a cell or cellular material is directly fixed to said solid support, and polynucleotide sequences within said material are hybridized to polynucleotide or oligonucleotide probes in situ.

27. The method according to claim 1 wherein said signalling moiety is a chemiluminescent agent.