Non-isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions useful therefor

• US 5,474,895 A
• Filed: 05/13/1993
• Issued: 12/12/1995
• Est. Priority Date: 11/14/1990
• Status: Expired due to Fees

• Current Assignee: Akzo Nobel N.V. (Nouryon BV)
• Original Assignee: Siska Diagnostics, Inc.
• Inventors: Ishii, Jennifer K., Ghosh, Soumitra
• Primary Examiner(s): Fleisher, Mindy B.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/050,441
• Time in Patent Office: 943 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 536/24.3, 536/24.31, 536/25.32
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form

Abstract

Methods for the detection of nucleic acid using sandwich hybridization are provided. The target nucleic acids are captured by hybridization with oligonucleotides covalently attached to polystyrene solid supports to form complexes which are then hybridized to detection oligonucleotides to facilitate the analysis of the target nucleic acid sequences.

Claims

1. A method for detecting a single-stranded target nucleic acid, comprising:

(a) hybridizing said target nucleic acid with a detection oligonucleotide to form a target nucleic acid-detection oligonucleotide complex,

wherein said detection oligonucleotide has a reporter enzyme covalently bound thereto and forms a stable hybrid with a first portion of the target nucleic acid;

(b) hybridizing said complex with a polystyrene support-bound capture oligonucleotide to form a sandwich complex comprising the capture oligonucleotide, the target nucleic acid, and the,

wherein the capture oligonucleotide is covalently bound to the polystyrene support and forms a stable hybrid with a second portion of said target nucleic acid, and said first and second portions do not overlap, and

wherein the hybridization reactions of steps (a) and (b) are conducted simultaneously or sequentially;

(c) washing said polystyrene support-bound, capture oligonucleotide-target nucleic acid-detection oligonucleotide sandwich complex under conditions sufficient to remove substantially all unhybridized detection oligonucleotide therefrom; and

(d) detecting said sandwich complexes, whereby the concentration of said target nucleic acid is measured.

2. The method of claim 1, wherein said detection is effected by reacting said sandwich complex with a suitable substrate under conditions such that the reporter enzyme promotes the conversion of said substrate to produce a detectable signal;

and measuring said signal and thereby determining the concentration of said target nucleic acid.

3. The method of claim 2, wherein said signal is detected by color metric, fluorescent, potentiometric, or chemiluminescent means and/or wherein said substrate is converted to a product which is detected by chemiluminescent means.

4. The method of claim 3, wherein said substrate is selected from the group consisting of dioxetane derivatized substrates for alkaline phosphatase, β

-galactosidase, aryl esterase and sulfatase.

5. The method of claim 2, wherein the reacting of sandwich complex with substrate is carried out at a temperature in the range of about 25°

-37°

C. for a time in the range of about 0.1 up to 5 hours.

6. The method of claim 1, wherein said target nucleic acid includes a sufficient number of nucleotides to form a stable hybrid with both said capture and detection oligonucleotides and is less than about 2000 nucleotides in length.

7. The method of claim 1, wherein said target nucleic acid is all or a detectable portion of a human immunodeficiency virus (HIV) genome.

8. The method of claim 1, wherein prior to the hybridization of step (a) said target nucleic acid is amplified.

9. The method of claim 8, wherein said amplification is effected by 3SR amplification, TAS amplification or PCR amplification.

10. The method of claim 1, wherein said polystyrene support is in the form of beads having a particle-size in the range of about 0.1 up to 10 microns, and wherein said. polystyrene beads contain a sufficient number of functional groups for covalent attachment of a concentration of at least 0.1 nmol per gram of polystyrene polymer of the capture oligonucleotide to the polystyrene polymer.

11. The method of claim 10, wherein said functional groups are selected from the group consisting of carboxyl groups, amine groups, aldehyde groups, hydrazine groups, epoxide groups, bromoacetyl groups, maleimide groups and thiol groups.

12. The method of claim 11, wherein the concentration of capture oligonucleotide, per gram of polystyrene polymer, falls in the range of about 1 up to 300 nmol/g.

13. The method of claim 1, wherein the capture oligonucleotide and/or detection oligonucleotide include at least about 20 nucleotides.

14. The method of claim 1, wherein the reporter enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, β

-galactosidase, aryl esterase, urease, and sulfatase.

15. The method of claim 1, wherein said hybridization of target nucleic acid with detection oligonucleotide is carried out at a temperature in the range of about 25°

-55°

C. for a time in the range of about 0,1 up to 6 hours.

16. The method of claim 15, wherein said hybridization is carried out in a buffer that contains a sufficient amount of glycerol to reduce the amount of denaturation of the reporter molecule and a concentration of salt in which hybrids that Include more than about 1% mismatched base pairs are unstable.

17. The method of claim 16, wherein the hybridization of target nucleic acid-detection oligonucleotide complex with capture oligonucleotide is carried out at a temperature in the range of about 25°

up to 55°

C. for about 0.1 up to 6 hours.

18. The method of claim 16, wherein said washing is effected by contacting said complex at a temperature in the range of about 25°

-50°

C. with a buffer containing in the range of about 0.1-2.0 X SSC and 0-0.1% SDS.

19. The method of claim 1, wherein the washing of the sandwich oligonucleotide complex is carried out under conditions suitable to remove substantially all non-specifically bound detection oligonucleotide and extraneous nucleic acids.

20. The method of claim 1, wherein the hybridization reactions of steps (a) and (b) are conducted sequentially.

21. A kit for the detection of target nucleic acids, comprising, in a container suitable for packaging, reagents for a bead-based sandwich hybridization assay for the detection of single-stranded, target nucleic acids, wherein said reagents include:

(a) a detection oligonucleotide that has a reporter enzyme covalently bound thereto and that includes a sufficient number of nucleotides that are complementary to a first portion of the target nucleic acid to form a stable hybrid;

(b) a capture oligonucleotide that is covalently bound to a polystyrene support and that includes a sufficient number of nucleotides that are complementary to a second portion of the target nucleic acid to form a stable hybrid, wherein said first and second portions do not overlap; and

(c) a substrate for the reporter enzyme, wherein said substrate is selected from the group of substrates converted by said enzyme into a product that is detectable by chemiluminescent, colorimetric, potentiometric, or fluorescent means.

22. The kit of claim 21, further comprising a container with a composition comprising hybridization buffer containing glycerol.