Assays

• US 10,167,525 B2
• Filed: 09/28/2014
• Issued: 01/01/2019
• Est. Priority Date: 11/06/2006
• Status: Expired due to Fees

• Current Assignee: Alere Technologies GmbH (Abbott Laboratories)
• Original Assignee: Alere Technologies GmbH (Abbott Laboratories)
• Inventors: Steinmetzer, Katrin, Ermantraut, Eugen, Schulz, Torsten, Kaiser, Thomas, Ullrich, Thomas
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US14/499,221
• Publication Number:
  US 20150197822A1
• Time in Patent Office: 1,556 Days
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/701 : Specific hybridization probes
• CPC Class Codes: C12Q 1/703 : Viruses associated with AIDS
• CPC Class Codes: C12Q 2600/158 : Expression markers

Abstract

A method includes forming a composition of matter including an amount of a reporter compound, a binding member capable of capturing the reporter compound, and an amount of a target nucleic acid capable of forming complexes with the reporter compound.

Claims

1. A method, comprising:

(a) providing;

an amount of a reporter compound;

a first binding member being configured to bind an anchor group of a capture molecule;

a second binding member capable of capturing the reporter compound;

an amount of a target nucleic acid capable of forming complexes with the reporter compound, the forming of complexes with the reporter compound inhibiting capturing of the reporter compound by the second binding member; and

an amount of capture molecules wherein each capture molecule comprises a binding portion specific to a region of the target nucleic acid and an anchor group;

(b) forming complexes each comprising the target nucleic acid and the capture molecule;

(c) contacting the complexes with the first binding member to bind the complexes to the first binding member

(d) releasing at least a subset of the amount of target nucleic acid from the first binding member;

(e) forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid;

(f) capturing a remaining subset of the amount of reporter compound not in complex with the target nucleic acid on the second binding member;

(g) determining a value indicative of the amount of reporter compound captured on the second binding member; and

(h) determining a value indicative of the amount of target nucleic acid based on the value indicative of the amount of reporter compound captured on the second binding member; and

(i) wherein the value indicative of the amount of reporter compound captured on the second binding member is determined before forming complexes of the subset of the amount of reporter compound with at least the subset of the amount of target nucleic acid.

2. The method of claim 1, further comprising determining a value indicative of the amount of target nucleic acid based on the value indicative of the amount of reporter compound captured on the binding member.

3. The method of claim 2, wherein the value indicative of the amount of target nucleic acid is determined based on a calibration curve correlating the value indicative of the presence and/or amount of reporter compound with the value indicative of the presence and/or amount of target nucleic acid.

4. The method of claim 1, further comprising releasing the remaining subset of the amount of reporter compound from the binding member after the steps of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid, capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the binding member, and determining the value indicative of the amount of reporter compound captured on the binding member.

5. The method of claim 4, further comprising performing the steps of releasing, forming complexes, capturing, and determining a number N additional times, where N is an integer greater than or equal to 1.

6. The method of claim 4, further comprising performing the steps of releasing, forming complexes, capturing, and determining a number N additional times, wherein N ≥

5 .

7. The method of claim 4, further comprising performing the steps of releasing, forming complexes, capturing, and determining a number N additional times, wherein N ≥

10 .

8. The method of claim 4, further comprising performing the steps of releasing, forming complexes, capturing, and determining a number N additional times, wherein N ≥

20 .

9. The method of claim 1, further comprising, prior to the step of forming complexes:

capturing at least a subset of the amount of reporter compound on the binding member;

determining a value indicative of the amount of reporter compound captured on the binding member; and

releasing captured reporter compounds from the binding member.

10. The method of claim 1, wherein the step of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid and the step of capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the binding member are performed concomitantly.

11. The method of claim 1, wherein the value indicative of the amount of reporter compound captured on the binding member is determined 1seconds to 120 seconds after initiating the steps of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid and of capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the binding member.

12. The method of claim 1, wherein a reporter compound comprises one or more detectable labels.

13. The method of claim 12, wherein the one or more detectable labels are fluorescent labels.

14. The method of claim 12, wherein the label is a compound that includes a chemical substance that generates a detectable signal in a physical reaction.

15. The method of claim 12, wherein the label is an enzyme that generates a detectable signal in an enzymatic reaction.

16. The method of claim 1, wherein the reporter compounds are oligonucleotides.

17. The method of claim 1, wherein forming a composition of matter comprises forming a composition of matter comprising:

an amount of a first reporter compound, an amount of a first target nucleic acid capable of forming complexes with the first reporter compound, the forming of complexes with the first reporter compound inhibiting capturing of the first reporter compound by the binding member, an amount of a second reporter compound, and an amount of a second target nucleic acid capable of forming complexes with the second reporter compound, the forming of complexes with the second reporter compound inhibiting capturing of the second reporter compound by the binding member.

18. The method of claim 1, wherein the binding member comprises one or more different capture molecules being capable of capturing a reporter compound on the binding member.

19. The method of claim 18, wherein the capture molecules are oligonucleotides.

20. The method of claim 18, wherein different capture molecules are arranged on different locations with respect to the binding member.

21. The method of claim 18, wherein the reporter compound is captured on the binding member by forming complexes with the capture molecules.

22. The method of claim 1, wherein determining the value indicative of the amount of reporter compound captured on the binding member comprises time-dependent monitoring of the indicative value.

23. The method of claim 1, further comprising subjecting the target nucleic acid to thermal cyclic amplification.

24. The method of claim 23, wherein the thermal cyclic amplification comprises at least 10 cycles.

25. The method of claim 23, wherein the thermal cyclic amplification comprises at least 20cycles.

26. The method of claim 23 wherein the thermal amplification is a PCR.

27. The method of claim 26, wherein performing the PCR comprises using a polymerase having exonuclease activity.