Methods and kits for performing nucleic acid hybridization assays

• US 4,751,177 A
• Filed: 06/13/1985
• Issued: 06/14/1988
• Est. Priority Date: 06/13/1985
• Status: Expired due to Term

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Stabinsky, Yitzhak
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/744,509
• Time in Patent Office: 1,097 Days
• Field of Search: 435/6, 435/810, 435/814, 436/501, 436/527, 436/531, 436/808, 436/824, 935/2, 935/3, 935/6, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/705 for herpetoviridae, e.g. herpes simplex, varicella zoster
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 930/224 Herpes related

Abstract

A method for the isolation and quantitative detection of a selected single-stranded target polynucleotide from solution. The target polynucleotide is hybridized in solution to a single-stranded mediator polynucleotide, a probe polynucleotide, and an immobilized polynucleotide sequence. The sequence of the mediator polynucleotide comprises a first sequence complementary to a first portion of the target polynucleotide sequence and a second nucleotide sequence complementary to a portion of a single-stranded immobilized polynucleotide sequence. The probe polynucleotide, which carries a detectable label, is complementary to a second portion of the necleotide sequence of the target. The immobilized polynucleotide is immobilized by attachment to a solid support, and, through hybridization to the mediator polynucleotide, functions to immobilize the entire immobilized polynucleotide/target polynucleotide/probe polynucleotide "sandwich".

Claims

1. A method for searching a sample solution for selected target polynucleotide suspended in the solution comprising the steps of:

(a) exposing the sample to a mediator polynucleotide having a first portion with a nucleotide sequence complementary to and hybridizable with an immobilized polynucleotide and having a second portion, separate and distinct from the first portion, with a nucleotide sequence complementary to and hybridizable with a first portion of the target polynucleotide;

(b) introducing to the sample a probe polynucleotide having an attached reporter group and having a portion with a nuceotide sequence complementary to and hybridizable with a second portion of the target polynucleotide, said second portion being separate and distinct from said first portion of the target polynucleotide;

(c) hybridizing the first portion of the target with the second portion ofthe mediator polynucleotide and the second portion of the target polynucleotide with the portion of the probe polynucleotide; and

(d) immobilizing the mediator polynucleotide by hybridization of the first portion of the mediator polynucleotide with the immobilized polynucleotide.

2. The method as recited in claim 1 further comprising the step of qualitating the amount of signal supplied by said detectable label on said immobilized hybrid product.

3. The method as recited in claim 1 wherein said target polynucleotide is a polydeoxyribonucleotide.

4. The method as recited in claim 1 wherein said target polynucleotide is a polyribonucleotide.

5. The method as recited in claim 3 or 4 wherein said mediator polynucleotide sequence is a deoxyribonucleic acid sequence.

6. The method as recited in claim 3 or 4 wherein said mediator polynucleotide sequence is ribonucleic acid sequence.

7. The method as recited in claim 3 or 4 wherein said probe polynucleotide sequence is a deoxyribonucleic acid sequence.

8. The method as recited in claim 3 or 4 wherein said probe polynucleotide sequence is a ribonucleic acid sequence.

9. The method as recited in claim 3 or 4 wherein said immobilized polynucleotide is a polydeoxyribonucleotide.

10. The method as recited in claim 3 or 4 wherein said immobilized nucleotide is a polyribonucleotide.

11. A kit for performing a hybridization assay on a sample to search for a selected target polynucleotide comprising:

a polynucleotide immobilized on a support;

a mediator polynucleotide, associated as a component of a kit with said immobilized polynucleotide, having a first portion with a nucleotide sequence complementary to and hybridizable with said immobilized polynucleotide and having a second portion, separate and distinct from the first portion, with a nucleotide sequence complementary to and hybridizable with a first portion of the target polynucleotide;

a polynucleotide probe, associated as a component of a kit with said immobilized polynucletide, having a portion with a nucleotide sequence complementary to and hybridizable with a second portion of the target polynucleotide, said second portion being separate and distinct from said first portion of the target polynucleotide; and

a reporter attached to said probe polynucleotide.

12. The kit as recited in claim 11 wherein said target polynucleotide is a polydeoxyribonucleotide.

13. The kit as recited in claim 11 wherein said target polynucleotide is a polyribonucleotide.

14. The kit as recited in claim 12 or 13 wherein said mediator polynucleotide sequence is a deoxyribonucleic acid sequence.

15. The kit as recited in claim 12 or 13 wherein said mediator polynucleotide sequence is a ribonucleic acid sequence.

16. The kit as recited in claim 12 or 13 wherein said probe polynucleotide sequence is a deoxyribonucleic acid sequence.

17. The kit as recited in claim 12 or 13 wherein said probe polynucleotide sequence is a ribonucleic acid sequence.

18. The kit as recited in claim 12 or 13 wherein said immobilized polynucleotide is a polydeoxyribonucleotide.

19. The kit as recited in claim 12 or 13 wherein said immobilized polynucleotide is a polyribonucleotide.