Digital assay

• US 20030165935A1
• Filed: 11/21/2002
• Published: 09/04/2003
• Est. Priority Date: 11/21/2001
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lao, Kai Q., Vann, Charles S.
• Application Number:
  US10/302,688
• Publication Number:
  US 20030165935A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

The invention relates to methods and compositions for the detection of targets in a sample.

Claims

1. A method for quantitating a target comprising;

forming a reaction mixture comprising;

a sample possibly containing the target;

a codeable label;

one or more target-specific probes, wherein each target-specific probe binds specifically to the target under selective binding conditions; and

a separating moiety;

treating the reaction mixture under reaction conditions such that a detectable complex is produced when the target is present, and wherein the detectable complex comprises the codeable label, the target-specific probe, and the separating moiety; and

quantitating the target by counting the number of codeable labels.

2. The method of claim 1, further comprising separating the detectable complex from codeable labels that are not included in the detectable complex after treating the reaction mixture and before quantitating the target.

3. A method for quantitating at least two different particular targets comprising;

forming a reaction mixture comprising;

a sample possibly containing two or more different particular targets;

a different codeable label specific for each different particular target;

one or more different target-specific probes specific for each different particular target that bind specifically to the target under selective binding conditions; and

a separating moiety;

treating the reaction mixture under reaction conditions such that when a particular target is present, a detectable complex is produced, which comprises the codeable label specific for the particular target, the target-specific probe specific for the particular target, and the separating moiety; and

quantitating each of the different particular targets by counting the number of codeable labels specific for each of the different particular targets.

4. The method of claim 3, further comprising separating any detectable complexes produced from codeable labels that are not included in the detectable complex after treating the reaction mixture and before quantitating each of the different particular targets.

7. The method of claim 4, wherein separating the ligation product from unligated detecting and separating beads comprises:

separating the ligation product from the target nucleic acid sequences, and

separating the ligation product from the sample.

8. The method of claim 7, wherein the separating of the ligation product from the target nucleic acid sequences comprises thermal denaturation.

9. The method of claim 8, further comprising removing any separating beads that are not in a ligation product prior to the quantitating the target nucleic acid sequences.

10. The method of claim 9, wherein the removing of any separating beads that are not in a ligation product comprises:

placing any separating beads and ligation products in a density gradient, wherein the separating beads and ligation products differ in density; and

removing any separating beads that are not in a ligation product.

5. A method for quantitating at least two different target nucleic acid sequences in a sample comprising:

forming a ligation reaction mixture by combining the sample with a different probe set specific for each of the at least two different target nucleic acid sequences, each probe set comprising (a) at least one separating bead, comprising a magnetic particle and a first target-specific probe, and (b) at least one detecting bead, comprising a codeable label, and a second target-specific probe;

wherein the target-specific probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;

subjecting the ligation reaction mixture to a ligation reaction, wherein adjacently hybridizing complementary target-specific probes are ligated to one another to form a ligation product comprising the separating bead and the detecting bead; and

quantitating each of the at least two different target nucleic acid sequences by counting the number of codeable labels.

6. The method of claim 5, further comprising separating any ligation product from unligated separating and detecting beads after treating the reaction mixture and before quantitating each of the at least two different target nucleic acid sequences.

11. The method of claim 5, wherein the codeable label has a level of intensity that is specific for the second target-specific probe.

12. The method of claim 11, wherein the separating bead further comprises a second codeable label, and wherein the second codeable label has a level of intensity that is specific for the first target-specific probe.

13. The method of claim 12, wherein each of the at least two probe sets that are specific for target nucleic acid sequences comprise codeable labels that have the same wavelength.

14. The method of claim 5, wherein the codeable label is one or more quantum dots.

15. The method of claim 14, wherein the detecting bead of each probe set comprises at least 1,000 quantum dots, wherein the quantum dots have predetermined wavelengths that make the detecting bead distinguishable from different detecting beads.

16. The method of claim 15, wherein the separating bead further comprises at least 1,000 quantum dots, wherein the quantum dots have predetermined wavelengths that make the separating bead distinguishable from different separating beads.

17. The method of claim 5, wherein the quantitating the at least two target nucleic acid sequences in the sample is performed in a detecting vessel comprising a groove on one surface of the detecting vessel near a magnetic source, wherein the separating bead fits in the groove, the detecting bead does not fit in the groove, and the ligation products attracted to the magnetic source are aligned.

18. The method of claim 5, wherein the ligation reaction mixture further comprises a ligation agent.

19. The method of claim 18, wherein the ligation agent is a ligase.

20. The method of claim 18, wherein the ligation agent is a thermostable ligase.

21. The method of claim 20, wherein the thermostable ligase is selected from at least one of Tth ligase, Taq ligase, and Pfu ligase.

22. The method of claim 5, wherein each separating bead differs in density from each detecting bead, such that the distance between any separating beads that are not in a ligation product and the ligation product allows attraction of the ligation product to a magnetic device and does not allow attraction of the separating beads that are not in a ligation product to the magnetic device.

23. The method of claim 22, wherein the quantitating the ligation product occurs in the presence of the sample.

24. A kit for detecting target nucleic acid sequences in a sample comprising:

a different bead set specific for each of the target nucleic acid sequences, the bead set comprising (a) at least one separating bead, comprising a magnetic particle, a first codeable label comprising two or more labels, and a first target-specific probe, wherein the first codeable label is specific for the first target-specific probe, and (b) at least one detecting bead, comprising a second codeable label comprising a set of two or more labels, and a second target-specific probe, wherein the second codeable label is specific for the second target-specific probe;

wherein the first codeable label is detectably different from the second codeable label; and

wherein the target-specific probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence.

25. The kit of claim 24, further comprising a ligation agent.

26. The kit of claim 25, wherein the ligation agent is a ligase.

27. The kit of claim 25, wherein the ligation agent is a thermostable ligase.

28. The kit of claim 27, wherein the thermostable ligase is selected from at least one of Tth ligase, Taq ligase, and Pfu ligase.

29. A method for detecting at least two different target nucleic acid sequences in a sample comprising:

forming a ligation reaction mixture by combining the sample with a different bead set specific for each of the at least two different target nucleic acid sequences, each bead set comprising (a) at least one separating bead, comprising a magnetic particle, a codeable label comprising at least two labels, and a first target-specific probe, wherein the first codeable label is specific for the first target-specific probe, and (b) at least one detecting bead, comprising a second codeable label comprising at least two labels and a second target-specific probe, wherein the second codeable label is specific for the second target-specific probe;

wherein the first codeable label is detectably different from the second codeable label;

wherein the target-specific probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;

subjecting the ligation reaction mixture to a ligation reaction, wherein adjacently hybridizing complementary target-specific probes are ligated to one another to form a detectable complex comprising the separating bead and the detecting bead; and

quantitating the at least two different target nucleic acid sequences in the sample by quantitating the detectable complex.

30. The method of claim 29, wherein the codeable labels comprise at least two phosphors.

31. The method of claim 29, wherein the codeable labels comprise at least two fluorescent molecules.

32. The method of claim 29, further comprising separating the detectable complex from unligated detecting and separating beads after the ligation reaction and prior to the quantitating the at least two different target nucleic acid sequences.

33. The method of claim 32, wherein separating the detectable complex from unligated detecting and separating beads comprises:

separating the detectable complex from the at least two different target nucleic acid sequences, and

separating the detectable complex from the sample.

34. The method of claim 33, wherein the separating of the detectable complex from the at least two different target nucleic acid sequences comprises thermal denaturation.

35. The method of claim 34, further comprising removing any separating beads that are not in a detectable complex prior to the quantitating the at least two different target nucleic acid sequences.

36. The method of claim 35, wherein the removing of any separating beads that are not in a detectable complex comprises:

placing any separating beads and detectable complexes in a density gradient, wherein the separating beads and detectable complexes differ in density; and

removing any separating beads that are not in a detectable complex.

37. The method of claim 29, wherein the detecting bead further comprises a magnetic particle.

38. The method of claim 37, wherein the quantitating the at least two target nucleic acid sequences in the sample is performed in a detection vessel comprising a groove on one surface of the detection vessel near a magnetic source, wherein the groove comprises a first end and a second end, and wherein the first codeable label and the second codeable label of the detectable complex are aligned within the groove with the magnetic source, such that the separating bead of the detectable complex aligns closer to the first end than the detecting bead of the detectable complex.