Solid phase amplification process

• US 6,017,738 A
• Filed: 12/09/1996
• Issued: 01/25/2000
• Est. Priority Date: 11/01/1991
• Status: Expired due to Fees

• Current Assignee: Diatech Pty Ltd.
• Original Assignee: Adelaide Children'S Hospital, University of South Australia (University of Adelaide)
• Inventors: Morris, Charles Phillip, Harris, Raymond John
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/761,862
• Time in Patent Office: 1,142 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

A method for detecting a target nucleic acid sequence comprises: (a) providing a first primer hybridizing to the target nucleic acid sequence, wherein the primer is immobilized on an immobile solid phase support by a direct chemical linkage between the primer and the solid phase support, wherein the solid phase support forms a part of or is insertable into a container for a sample to be tested, (b) providing a second primer hybridizing to the target nucleic acid sequence in the opposite direction, wherein the second primer is labelled with a detectable label, (c) reacting the first and second primers with a sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, and (d) detecting the presence of bound second primer. Alternatively, the label on the second primer can be attached or incorporated either during or after the amplification process. An assay system or kit for use in this method includes a first primer hybridizing to the target nucleic acid sequence, a second primer hybridizing to the target nucleic acid sequence in the opposite direction, and reagents for amplification of the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, and reagents for detection of the label on the bound second primer.

Claims

1. A method for detecting a target nucleic acid sequence by a single-stage amplification process conduct ed within a single container, said method comprising:

(a) conducting, within a single container, an amplification process having only a single stage comprising the following steps;

(i) providing a first primer hybridizing to the target nucleic acid sequence, wherein the first primer is immobilized on an immobile solid phase support by a direct chemical linkage between the first primer and the solid phase support, wherein the solid phase support forms a part of or is insertable into a container for a sample to be tested,

(ii) providing a second primer hybridizing to the target nucleic acid sequence in the opposite direction, wherein the second primer is labeled with a detectable label,

(iii) adding an aliquot of the first primer or other primer(s) to a sample solution containing nucleic acid sequences, in the container together with said first primer of step (i) and said second primer, and

(iv) reacting the first, second and other primer(s) with the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first, second and other primers in the container for the sample, and

(b) within the same container, detecting the presence of bound second primer.

2. The method of claim 1 wherein the container is a microtiter plate well.

3. The method of claim 1 wherein the first primer is immobilized on a solid phase support which is immersible into the container.

4. The method of claim 1 wherein the nucleic acid sequence is amplified by the polymerase chain reaction.

5. The method of claim 1 wherein the nucleic acid sequence is amplified by the nucleic acid sequence based amplification.

6. The method of claim 1 wherein the nucleic acid sequence is amplified by the ligase chain reaction based amplification.

7. The method of claim 1 wherein the nucleic acid sequence is amplified by the Qβ

replicase based amplification.

8. The method of claim 1 wherein the nucleic acid sequence is amplified by the strand displacement amplification.

9. The method of claim 1, wherein the nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of organisms, nucleic acid sequences characteristic of a genetic disorder or disease, nucleic acid sequences characteristic of HLA types, nucleic acid sequences which serve to identify an organism, and nucleic acid sequences characteristic of the sex of an organism.

10. The method of claim 1 wherein the label is selected from the group consisting of fluorescent labels, biotin, radiolabels, enzymes specific for chromogenic substrates and dyes.

11. An assay system or kit for detecting a target nucleic acid sequence by a method according to claim 1, said assay system or kit comprising:

a single container for a sample to be tested,

a first primer hybridizing to the target nucleic acid sequence, wherein the first primer is immobilized on an immobile solid phase support by a direct chemical linkage between the primer and the solid phase support,

wherein the solid phase support forms a part of or is insertable into said container for the sample to be tested,

a second primer hybridizing to the target nucleic acid sequence in the opposite direction,

wherein the second primer is labelled with a detectable molecule,

an aliquot of the first primer, unattached to the immobile solid phase support, and

reagents for amplification of the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, wherein said aliquot modifies the amplification of said target sequence by enabling solid phase and solution phase amplification to take place concurrently, and

reagents for detection of the label on the bound second primer.

12. The assay system or kit of claim 11 wherein the container is a microtiter plate well, further comprising the well plate.

13. The assay system or kit of claim 11 wherein the first primer is immobilized on an immobile solid phase support which is immersible into the container.

14. The assay system or kit of claim 11 wherein the reagents for amplifying the nucleic acid sequence are those for use in the polymerase chain reaction.

15. The assay system or kit of claim 11 wherein the reagents for amplifying the nucleic acid sequence are those for use in the nucleic acid sequence based amplification.

16. The assay system or kit of claim 11 wherein the reagents for amplifying the nucleic acid sequence are those for use in the ligase chain reaction based amplification.

17. The assay system or kit of claim 11 wherein the reagents for amplifying the nucleic acid sequence are those for use in the Qβ

replicase based amplification.

18. The assay system or kit of claim 11 wherein the reagents for amplifying the nucleic acid sequence are those for use in the strand displacement amplification process.

19. The assay system or kit of claim 11, wherein the nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of organisms, nucleic acid sequences characteristic of a genetic disorder or disease, nucleic acid sequences characteristic of HLA types, nucleic acid sequences which serve to identify an organism, and nucleic acid sequences characteristic of the sex of an organism.

22. An assay system or kit for detecting a target nucleic acid sequence by a method according to claim 1, said assay system or kit comprising:

a single container for a sample to be tested,

a first primer hybridizing to the target nucleic acid sequence, wherein the first primer is immobilized on an immobile solid phase support by a direct chemical linkage between the primer and the solid phase support,

wherein the solid phase support forms a part of or is insertable into said container for the sample to be tested,

a second primer hybridizing to the target nucleic acid sequence in the opposite direction,

wherein the second primer is labelled with a detectable molecule,

amplification means for modifying solid phase amplification which comprises an aliquot of the first primer, unattached to the immobile solid phase support, and

reagents for amplification of the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, wherein said amplification means enables solid phase and solution phase amplification to take place concurrently, and

reagents for detection of the label on the bound second primer.

20. A method for detecting a target nucleic acid sequence by a single-stage amplification process conducted within a single container, said method comprising:

(a) conducting, within a single container, an amplification process having only a single stage comprising the following steps;

(i) providing a first primer hybridizing to the target nucleic acid sequence, wherein the first primer is immobilized on an immobile solid phase support by a direct chemical linkage between the first primer and the solid phase support, wherein the solid phase support forms a part of or is insertable into a container for a sample to be tested,

(ii) providing a second primer hybridizing to the target nucleic acid sequence in the opposite direction,

(iii) adding an aliquot of the first primer or other primer(s) to a sample solution containing nucleic acid sequences, in the container together with said first primer of step (i) and said second primer, and

(iv) reacting the first, second and other primers with the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first, second and other primers in the container for the sample and, during the amplification process, labelling the second primer by attaching or incorporating a detectable label, and

(b) within the same container, detecting the presence of bound second primer.

21. A method for detecting a target nucleic acid sequence by a single-stage amplification process conducted within a single container, said method comprising:

(a) conducting, within a single container, an amplification process having only a single stage comprising the following steps;

(i) providing a first primer hybridizing to the target nucleic acid sequence, wherein the first primer is immobilized on an immobile solid phase support by a direct chemical linkage between the first primer and the solid phase support, wherein the solid phase support forms a part of or is insertable into a container for a sample to be tested,

(ii) providing a second primer hybridizing to the target nucleic acid sequence in the opposite direction,

(iii) adding an aliquot of the first primer or other primer(s) to a sample solution containing nucleic acid sequences, in the container together with said first primer of step (i) and said second primer, and

(iv) reacting the first, second and other primers with the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first, second and other primers in the container for the sample, and

(b) within the same container,

(i) after the amplification process, labelling the second primer by attaching or incorporating a detectable label, and

(ii) detecting the presence of bound second primer.