Detection of DNA or RNA using single molecule arrays and other techniques

• US 10,640,814 B2
• Filed: 03/30/2018
• Issued: 05/05/2020
• 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
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/942,379
• Publication Number:
  US 20180363038A1
• Time in Patent Office: 767 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 sample, comprising:

exposing DNA or RNA fragments derived from DNA or RNA molecules from a sample, each having a sequence, to at least a plurality of first capture objects and a plurality of second capture objects, wherein at least a portion of the DNA or RNA fragments associate with at least one of the first capture objects and the second capture objects, wherein each of the first capture objects comprises a first capture probe comprising a first nucleic acid sequence complimentary to a first portion of the sequence of the DNA or RNA molecules, and each of the second capture objects comprises a second capture probe comprising a second nucleic acid sequence complimentary to a second portion of the sequence of the DNA or RNA molecules, wherein the first portion and the second portion of the sequence are different, and wherein the first capture objects and the second capture objects produce substantially similar signals; and

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

2. The method of claim 1, wherein the exposing comprises:

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

3. The method of claim 2, wherein the number of DNA or RNA fragments associated with at least one of the first capture objects and the second capture objects is determined by determining the number of locations containing at least one of the first capture objects and the second capture objects associated with a DNA or RNA fragment.

4. The method of claim 2, wherein the plurality of locations comprises a plurality of reaction vessels.

5. The method of claim 4, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters.

6. The method of claim 4, wherein the average volume of the plurality of reaction vessels is between about 1 femtoliter and about 1 picoliter.

7. The method of claim 4, further comprising sealing the plurality of reaction vessels.

8. The method of claim 2, wherein in the analyzing step, the number of locations containing at least one of the first capture objects and the second capture objects but not associated with any DNA or RNA fragment or a binding ligand is determined.

9. The method of claim 2, wherein the plurality of locations is addressed using optical techniques.

10. The method claim 1, wherein the exposing 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 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.

11. The method of claim 1, further comprising exposing the DNA or RNA fragments, at least some of which are associated with a capture object, to a plurality of binding ligands, wherein at least some of the DNA or RNA fragments associate with at least one binding ligand.

12. The method of claim 11, wherein in the analyzing step, the number of said locations containing a capture object that includes a binding ligand is determined.

13. The method of claim 11, wherein the number of locations comprising a DNA or RNA fragment is determined by determining the number of locations containing a binding ligand.

14. The method of claim 13, 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.

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

16. The method of claim 15, wherein the measure of the concentration of DNA or RNA molecules in the sample is based at least in part on the number of locations determined to contain a DNA or RNA fragment.

17. The method of claim 15, wherein the measure of the concentration of DNA or RNA molecules in the sample is determined at least in part by comparison of a measured parameter to a calibration standard.

18. The method of claim 1, wherein the first capture objects and the second capture objects produce signals comprising emission of electromagnetic energy differing in wavelength by not more than 50 nm.

19. The method of claim 1, wherein the first capture objects and the second capture objects produce signals comprising emission of electromagnetic energy differing in wavelength by not more than 10 nm.

20. The method of claim 1, wherein the first capture objects and the second capture objects produce indistinguishable signals.

21. The method of claim 1, further comprising fragmenting DNA or RNA molecules from the sample to form the DNA or RNA fragments.

22. The method of claim 1, wherein at least a portion of the DNA or RNA fragments associate with at least one of the first capture objects, and at least a portion of the DNA or RNA fragments associate with at least one of the second capture objects.

23. The method of claim 1, wherein the first capture objects and the second capture objects comprise beads.

24. The method of claim 1, further comprising performing at least one wash step.