Detection of plurality of targets in biological samples

• US 9,677,125 B2
• Filed: 10/21/2009
• Issued: 06/13/2017
• Est. Priority Date: 10/21/2009
• Status: Active Grant

• Current Assignee: Leica Microsystems CMS GmbH (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Gerdes, Michael John, Sood, Anup, Nelson, John Richard
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US12/582,745
• Publication Number:
  US 20110092381A1
• Time in Patent Office: 2,792 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

Methods for detecting a plurality of targets in a biological sample are provided. The method comprises contacting the biological sample with a plurality of target-binding probes to form a plurality of target-bound probes, covalently attaching at least one of the target-bound probes to the biological sample, and observing the signals from the target-bound probes sequentially. An associated kit and device for detection of the plurality of targets are also provided.

Claims

1. A method of detecting at least three sets of different targets in a biological sample via simultaneous addition of at least three sets of different target-binding probes followed by their sequential detection, the method comprising:

(a) simultaneously contacting the at least three sets of different targets with the at least three sets of different target-binding probes to form a plurality of target-bound probes;

(b) covalently attaching each of the plurality of target-bound probes of step (a) to the corresponding target or to a molecule that is located in the vicinity of the corresponding target in the biological sample to form a plurality of covalently attached probes; and

(c) sequentially detecting the plurality of covalently attached probes of step (b) to sequentially detect each set of different target, wherein the sequential detection comprises;

(i) generating a first set of signals from a first set of probes in the plurality of covalently attached probes by contacting a first set of signal-generating probes that are capable of binding to the first set of probes;

(ii) detecting a first set of targets via observing the first set of signals that is generated in step (i);

(iii) modifying the observed first set of signals by applying a chemical agent after step (ii);

(iv) generating a second set of signals after step (iii) from a second set of probes in the plurality of covalently attached probes by contacting a second set of signal-generating probes that are capable of binding to the second set of probes;

(v) detecting a second set of targets via observing the second set of signals that is generated in step (iv);

(vi) modifying the observed second set of signals by applying a chemical agent after step (v); and

(vii) generating a subsequent set of signals from a subsequent set of probes in the plurality of covalently attached probes, detecting a subsequent set of targets via observing the subsequent set of signals, modifying the observed subsequent set of signals by applying a chemical agent, and repeating these steps till all sets of different targets are detected.

2. The method of claim 1, wherein the signal generation, the target detection via signal observation and the signal modification steps in step (vii) are performed on a third, a fourth, and an n^th set of the plurality of covalently attached probes using a third, a fourth, and an n^th set of signal-generating probes to observe a third, a fourth, and an nth set of signals and thereby to detect a third, a fourth, and an nth set of targets, wherein integer value of n ranges from 5 to 100.

3. The method of claim 1, further comprising imaging the biological sample to obtain a background signal signature prior to generating the first set of signals.

4. The method claim 3, further comprising subtracting the background signal signature from the signals observed in the subsequent signal observing steps.

5. The method of claim 1, further comprising steps for removing the target-binding probes that are not bound to the targets prior to step (b).

6. The method of claim 5, further comprising steps for removing the first set of signal-generating probes that are not bound to the first set of probes in the plurality of covalently attached probes, prior to detecting the first set of targets.

7. The method of claim 1, wherein the targets are selected from the group consisting of a deoxyribonucleic acid, a ribonucleic acid, a protein, and a combination thereof.

8. The method of claim 1, wherein the at least three sets of different targets consist essentially of at least three sets of different ribonucleic acids.

9. The method of claim 1, wherein at least four sets of different targets are detected by simultaneously contacting with at least four sets of different target-binding probes.

10. The method of claim 1, wherein the chemical agent is selected from the group consisting of an acid, a base, a nucleophile, an electrophile, an oxidizing agent, a reducing agent and combinations thereof.

11. The method of claim 1, wherein the first set of signal-generating probes, the second set of signal-generating probes and the subsequent set of signal-generating probes are cyanine dyes.