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

• US 9,932,626 B2
• Filed: 01/15/2014
• Issued: 04/03/2018
• 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
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/761,271
• Publication Number:
  US 20150353997A1
• Time in Patent Office: 1,539 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 a measure of 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, at least a portion of the fragmented DNA or RNA associates with at least one of the second type of binding ligand, and at least a portion of the fragmented DNA or RNA associates with at least one of the first type of binding ligand and 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 a total number of fragmented DNA or RNA sequences associated with (i) the first type of binding ligand, and (ii) the second type of binding ligand, and (iii) both 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; and

determining a measure of the concentration of the DNA or RNA molecules in the sample based on the total number of fragmented DNA or RNA sequences determined in the analyzing step.

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.

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 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.

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

7. The method of claim 6, wherein the average volume of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters.

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

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

10. The method of claim 9, wherein the precursor labeling agent is converted to a labeling agent upon exposure to a secondary binding ligand.

11. The method claim 10, 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.

12. The method of claim 10, wherein the number of locations comprising a fragmented DNA or RNA sequence is determined by determining the number of locations containing a labeling agent.

13. The method of claim 10, wherein the precursor labeling agent is a enzymatic substrate.

14. 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.

15. The method of claim 14, wherein the moiety comprises biotin.

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

17. 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, at least one of the second type of binding ligand, or at least one of the first type of binding ligand and at least one of the second type of binding ligand to a plurality of secondary binding ligands, wherein a portion of each of the first type of binding ligands and the second type of binding ligands associated with the DNA or RNA sequences associates with a secondary binding ligand.

18. The method of claim 17, wherein the secondary binding ligand comprises an enzymatic component.

19. The method of claim 17, 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 one, two, or all three 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 of claim 1, wherein the first type of binding ligand and the second type of binding ligand each produce signals comprising emission of electromagnetic energy differing from each other in wavelength by not more than 10 nm.

21. 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.

22. The method of claim 21, wherein the immobilizing is carried out prior to the exposing step.

23. The method of claim 21, wherein a portion of the capture objects subjected to the immobilizing step are spatially segregated into a plurality of separate locations.

24. The method of claim 21, wherein the plurality of capture objects comprises a plurality of beads.

25. The method of claim 21, wherein each capture object includes a binding surface having affinity of at least one DNA or RNA fragment.