DETECTION OF DNA OR RNA USING SINGLE MOLECULE ARRAYS AND OTHER TECHNIQUES

• US 20180363038A1
• Filed: 03/30/2018
• Published: 12/20/2018
• Est. Priority Date: 01/15/2013
• Status: Active Grant

• Current Assignee: Quanterix Corporation
• Original Assignee: Quanterix Corporation
• Inventors: Duffy, David C., Song, Linan, Shan, Dandan, Zhao, Mingwei
• Granted Patent: US 10,640,814 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label

Abstract

Described herein are methods and systems for detecting DNA or RNA using single molecules array or other techniques. DNA or RNA from the sample may be fragmented and exposed to a first type of binding ligand and a second type of binding ligand that comprise nucleic acid sequences complimentary at least a portion of a sequence contained in the target DNA or RNA. At least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand and/or the second type of binding ligand, wherein the first type of binding ligand and second type of binding ligand comprises nucleic acid sequences complimentary to a different portions of a sequence contained in the DNA or RNA. A portion of the sample exposed to the binding ligands is analyzed to determine the number of fragmented DNA or RNA sequences.

Claims

1. A method for determining detecting or measuring the concentration of DNA or RNA molecules in a sample, comprising:

fragmenting DNA or RNA from the sample to form fragmented DNA or RNA sequences;

exposing the fragmented DNA or RNA sequences to at least a first type of binding ligand and a second type of binding ligand, wherein at least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand and/or the second type of binding ligand, wherein the first type of binding ligand comprises a first nucleic acid sequence complimentary to a first portion of a sequence contained in the DNA or RNA and second type of binding ligand comprises a second nucleic acid sequence complimentary to a second portion of a sequence contained in the DNA or RNA, wherein the first nucleic acid sequence and the second nucleic acid sequence are different; and

analyzing a portion of the sample from the exposing step to determine the number of fragmented DNA or RNA sequences associated at least one of the first type of binding ligand and the second type of binding ligand, wherein the first type of binding ligand and the second type of binding ligand produce substantially similar signals.

2. The method of claim 1, wherein the first type of binding ligand and the second type of binding ligand produce signals comprising emission of electromagnetic energy differing in wavelength by not more than 50 nm, optionally by not more than 40 nm, optionally by not more than 30 nm, optionally by not more than 20 nm, optionally by not more than 10 nm, optionally by not more than 5 nm, and optionally by not more than 1 nm.

3. The method of claim 1, wherein the first type of binding ligand and the second type of binding ligand produce indistinguishable signals.

4. The method of any preceding claim 1, wherein the exposing further comprises:

spatially segregating at least a portion of the DNA or RNA fragments into a plurality of separate locations.

5. The method claim 4, wherein the number of fragmented DNA or RNA sequences is determined by determining the number of locations containing a DNA or RNA fragment.

7. The method of claim 4, wherein the plurality of locations comprises a plurality of reaction vessels.

8. The method of claim 7, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters, or between about 1 femtoliter and about 1 picoliter.

9. The method of claim 1, wherein each of the least one of the first type of binding ligand and each of the at least one second type of binding ligand comprise a moiety which is capable of binding a secondary binding ligand.

12. The method of claim 1, further comprising exposing the fragmented DNA or RNA sequences associated with at least one of the first type of binding ligand or the second type of binding ligand to a plurality of secondary binding ligands, wherein a portion of each of the at least one first type of binding ligand and the at least one second type of binding ligand associated with the DNA or RNA sequences associates with a secondary binding ligand.

14. The method of claim 4, wherein following the spatially segregating step, the locations are exposed to precursor labeling agent.

15. The method of claim 14, wherein the precursor labeling agent is converted to a labeling agent upon exposure to a secondary binding ligand.

16. The method claim 15, wherein the number of locations comprising a fragmented DNA or RNA sequence is determined by determining the number of locations containing a secondary binding ligand.

19. The method of claim 12, further comprising exposing the fragmented DNA or RNA sequences to a third type of binding ligand, wherein at least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand, the second type of binding ligand, or the third type of binding ligand, wherein the third type of binding ligand comprises a third nucleic acid sequence complimentary to a third portion of a sequence contained in the DNA or RNA, wherein the first, second, and third nucleic acid sequences are different.

20. The method claim 19, further comprising exposing the fragmented DNA or RNA sequences to a fourth type of binding ligand, wherein at least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand, the second type of binding ligand, the third type of binding ligand, and/or the fourth type of binding ligand, wherein the fourth type of binding ligand comprises a fourth nucleic acid sequence complimentary to a fourth portion of a sequence contained in the DNA or RNA, wherein the first, second, third, and fourth nucleic acid sequences are different.

21. The method claim 20, further comprising exposing the fragmented DNA or RNA sequences to a fifth type of binding ligand, wherein at least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand, the second type of binding ligand, the third type of binding ligand, the fourth type of binding ligand, and/or the fifth type of binding ligand, wherein the fifth type of binding ligand comprises a fifth nucleic acid sequence complimentary to a fifth portion of a sequence contained in the DNA or RNA, wherein the first, second, third, fourth, and fifth nucleic acid sequences are different.

22. The method of claim 1, further comprising determining a measure of the concentration of DNA or RNA molecules in the sample.

23. The method of claim 4, wherein the measure of the concentration of DNA or RNA molecule in the sample based at least in part on the number of locations determined to contain a DNA or RNA fragments.

24. The method of claim 1, wherein the method further comprises immobilizing the DNA or RNA fragments with respect to a plurality of capture objects such that at least some of the capture objects associate with at least one DNA or RNA fragment and a statistically significant fraction of the capture objects do not associate with any DNA or RNA fragments.

25. The method of claim 24, wherein the immobilizing is carried out prior to the exposing step.

26. The method of claim 24, wherein a portion of the capture objects subjected to the immobilizing step are spatially segregated into a plurality of separate locations.

27. The method of claim 24, wherein each capture objects includes a binding surface having affinity for at least one DNA or RNA fragment.

28. The method claim 27, wherein the binding surface comprises a plurality of capture probes.

29. The method of claim 28, wherein the plurality of capture probes comprises a plurality of types of capture probes, each type of capture probe comprising a nucleic acid sequence complimentary to differing portions of a sequence contained in the DNA or RNA molecule.

30. The method of claim 27, wherein in the analyzing step, the number of locations containing a capture object that includes a binding surface having affinity for at least one type of DNA or RNA fragment but not containing a DNA or RNA fragment or a binding ligand is determined.

31. The method of claim 30, wherein the measure of the concentration of DNA or RNA molecules in the sample is based at least in part on the fraction of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of DNA or RNA fragment which contains a DNA or RNA fragment or binding ligand, to the total number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of DNA or RNA fragment.

37. The method of claim 1, wherein the first type of binding ligand and the second type of binding ligand produce indistinguishable signals

6. (canceled)

10-11. -11. (canceled)

13. (canceled)

17-18. -18. (canceled)

32-36. -36. (canceled)

38. A method for determining detecting or measuring the concentration of DNA or RNA molecules in sample, comprising:

fragmenting DNA or RNA from a sample to form fragmented DNA or RNA sequences;

exposing the fragmented DNA or RNA sequences to at least a first type of capture object and a second type of capture object, wherein at least a portion of the fragmented DNA or RNA associates with the least one of the first type of capture object and the second type of capture object, wherein the first type of capture object comprises a first capture probe comprising a first nucleic acid sequence complimentary to a portion of a sequence contained in the DNA or RNA and the second type of capture object comprises a second capture probe comprising a second nucleic acid sequence complimentary to a portion of a sequence contained in the DNA or RNA, wherein the first sequence and the second sequence are different; and

analyzing a portion of the sample from the exposing step to determine the number of fragmented DNA or RNA sequences associated with at least one of the first type of capture object and the second type of capture object.

39. The method of claim 38, wherein the exposing comprises:

spatially segregating at least a portion of the DNA or RNA fragments into a plurality of separate locations.

40. The method claim 38, wherein the exposing comprises:

immobilizing the DNA/RNA fragments with respect to a plurality of capture objects such that at least some of the capture objects associate with at least one DNA or RNA fragments and a statistically significant fraction of the capture objects do not associate with any DNA or RNA fragments;

spatially segregating at least a portion of the capture objects subjected to the immobilizing step into a plurality of separate locations.

41. The method of claim 39, wherein the number of fragmented DNA or RNA sequences associated at least one of the first type of capture object and the second type of capture object is determined by determining the number of locations containing at least one of the first type of capture object and the second type of capture object associated with a fragmented DNA or RNA sequence.

42. The method of claim 39, wherein the plurality of locations comprises a plurality of reaction vessels.

43. The method of claim 42, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters, or between about 1 femtoliter and about 1 picoliter.

44. The method of claim 38, further comprising exposing the fragmented DNA or RNA, at least some of which is associated with a capture object, to a plurality of binding ligands, wherein at least some of the fragmented DNA or RNA associates with at least one binding ligand.

45. The method of claim 44, wherein in the analyzing step, the number of said locations containing a capture object that includes a binding ligand is determined.

46. The method of claim 44, wherein the number of locations comprising a fragmented DNA or RNA sequence is determined by determining the number of locations containing a binding ligand.

47. The method of claim 38, further comprising determining the a measure of the concentration of DNA or RNA molecules in the sample.

48. The method of claim 47, wherein the measure of the concentration of DNA or RNA molecule in the sample based at least in part on the number of locations determined to contain a DNA or RNA fragments.

49. The method of claim 39, wherein in the analyzing step, the number of locations containing at least one of the first type of capture object and the second type of capture object but not associated with any DNA or RNA fragment or a binding ligand is determined.

50. The method of claim 46, wherein the measure of the concentration of DNA or RNA molecules in the sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of DNA or RNA fragment containing a DNA or RNA fragment or binding ligand, to the total number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of DNA or RNA fragment.

56. The method of claim 38, wherein the first type of capture object and the second type of capture object produce substantially similar signals

57. The method of claim 56, wherein the first type of capture object and the second type of capture object produce signals comprising emission of electromagnetic energy differing in wavelength by not more than 50 nm, optionally by not more than 40 nm, optionally by not more than 30 nm, optionally by not more than 20 nm, optionally by not more than 10 nm, optionally by not more than 5 nm, and optionally by not more than 1 nm.

51-55. -55. (canceled)