Method and reagent for detecting multiple nucleic acid sequences in a test sample

• US 5,955,268 A
• Filed: 04/26/1996
• Issued: 09/21/1999
• Est. Priority Date: 04/26/1996
• Status: Expired due to Term

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Carrino, John J., Granados, Edward N., Solomon, Natalie A., Bouma, Stanley R.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/639,224
• Time in Patent Office: 1,243 Days
• Field of Search: 435/6, 435/91, 435/194, 435/91.2, 435/91.5, 435/91.51, 536/22.1, 536/24.3, 536/24.33, 935/6, 935/17, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

Provided herein are methods for detecting multiple target nucleic acid sequences in a test sample. Also provided is a hybridization platform useful for detecting multiple target sequences in a test sample. The hybridization platform comprises a solid support material having a defined pattern of capture probes immobilized thereon.

Claims

1. A method of detecting a plurality of target sequences in a test sample comprising the steps of:

a) amplifying said target sequences in said test sample to form amplified target sequences;

b) contacting a hybridization platform with said amplified target sequences wherein

(i) said hybridization platform comprises at least two capture probes immobilized to a support material in a defined pattern,

(ii) said at least two capture probes have distinct sequences, and

(iii) each of said capture probes is labeled with at least one member of a signal generating system wherein said signal generating system generates a change in signal in a target dependent manner;

b) hybridizing at least two amplified target sequences to said hybridization platform to generate a change in signal at a site where said target sequence is hybridized; and

c) detecting a change in signal at said site as an indication of the presence of said target sequence in said test sample.

2. The method of claim 1 wherein said capture probes are labeled with an intercalation dye and said change in signal is detected upon hybridization of said target sequences to said hybridization platform.

3. The method of claim 1 wherein said probes are labeled with a dye that changes its fluorescence characteristics when in proximity of another dye and said method further comprises the step of labeling said target sequences with a dye that changes its fluorescence characteristics when in proximity of another dye before or after hybridizing said at least two target sequences to said hybridization platform but before detecting said change in signal.

4. The method of claim 1 wherein said capture probes are labeled with a quenching group and a reporter group whereby said signal generating group is permanently immobilized to said support material and said method further comprises degrading said capture probes in a target dependent manner after hybridizing said target to said platform but before detecting said change in signal.

5. The method of claim 4 wherein degrading said capture probes comprises contacting said probes with an enzyme having an activity selected from the group consisting of:

RNase H activity, restriction activity, endonuclease IV activity, endonuclease activity and exonuclease III activity.

6. A method of detecting a plurality of target sequences in a test sample comprising the steps of:

a) contacting a hybridization platform with said test sample comprising at least two target sequences wherein

(i) said hybridization platform comprises at least two capture probes immobilized to a support material in a defined pattern,

(ii) said at least two capture probes have distinct sequences, and

(iii) each of said capture probes is labeled with a quenching group and a reporter group whereby the signal generating group is permanently immobilized to said support material;

b) hybridizing at least two target sequences to said hybridization platform and degrading said capture probes in a target dependent manner to generate a change in signal at a site where said target sequence is hybridized; and

c) detecting a change in signal at said site as an indication of the presence of said target sequence in said test sample.

7. The method of claim 6 wherein said reagents for performing an amplification reaction comprise:

a) an enzyme having nuclease or exonuclease activity and polymerase activity; and

b) nucleotide triphosphates.

8. The method of claim 7 wherein said capture probes further comprise means for stopping the degradation of said capture probes.

9. A kit comprising reagents for performing an amplification reaction and a hybridization platform comprising at least two capture probes immobilized to a solid support material wherein

a) said capture probes have distinct sequences,

b) each of said capture probes is labeled with at least one member of a signal generating system wherein said signal generating system generates a change in signal in a target dependent manner, and

c) said capture probes are immobilized to said solid support material in a defined pattern.

10. The kit platform of claim 9 wherein said capture probes are labeled with a reporter group and a quenching group such that said signal generating group is permanently immobilized to said solid support material.

11. The kit platform of claim 10 wherein said capture probes comprise means for stopping capture probe degradation between said reporter group and said solid phase.