ENZYME-INDEPENDENT MOLECULAR INDEXING

• US 20180037942A1
• Filed: 08/01/2017
• Published: 02/08/2018
• Est. Priority Date: 08/03/2016
• Status: Abandoned Application

• Current Assignee: Cellular Research, Inc. (Becton Dickinson & Co)
• Original Assignee: Cellular Research, Inc. (Becton Dickinson & Co)
• Inventors: Fu, Glenn
• Application Number:
  US15/665,906
• Publication Number:
  US 20180037942A1
• 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 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present disclosure relates to compositions, methods and kits for quantitative analysis of a plurality of nucleic acid target molecules in a sample. In some embodiments, the methods comprise associating molecular label sequences with target nucleic acid molecules without an enzymatic reaction.

Claims

1. A method of quantitative analysis of a plurality of nucleic acid target molecules in a sample comprising:

providing a sample comprising a plurality of nucleic acid target molecules;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target specific region, a molecular label sequence, and a binding site for a first universal primer, wherein the molecular label sequence is selected from a diverse set of unique molecular label sequences, and wherein the molecular label sequences of two of the plurality of oligonucleotide probes are different;

contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization;

removing oligonucleotide probes that are not hybridized to the plurality of nucleic acid target molecules;

amplifying oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules using the first universal primer to generate a plurality of amplicons, wherein each of the plurality of amplicons comprises a target specific region and a molecular label sequence; and

determining the number of unique molecular label sequences for each target specific region,

whereby the quantity of each nucleic acid target molecule in the sample is determined.

2. The method of claim 1, further comprising immobilizing the plurality of nucleic acid target molecules on a solid support via an affinity moiety and a binding partner of the affinity moiety.

3. The method of claim 2, wherein the plurality of nucleic acid target molecules comprises the affinity moiety.

4. The method of claim 2 or 3, wherein the affinity moiety comprises a functional group of biotin, streptavidin, heparin, an aptamer, a click-chemistry moiety, digoxigenin, primary amine, carboxyl, hydroxyl, aldehyde, ketone, or any combination thereof.

5. The method of claim 4, wherein the plurality of nucleic acid target molecules is biotinylated.

6. The method of claim 4, wherein the plurality of nucleic acid target molecules is hybridized to a plurality of biotinylated capture probes.

7. The method of claim 6, wherein each of the plurality of biotinylated capture probes comprises a second target specific region.

8. The method of claim 7, wherein the second target specific region comprises poly dT.

9. The method of any one of claims 3-8, wherein the solid support comprises the binding partner of the affinity moiety.

10. The method of any one of claims 2-9, wherein removing oligonucleotide probes that are not hybridized to the plurality of nucleic acid target molecules comprises washing the solid support.

11. The method of any one of claims 1-10, wherein each of the plurality of oligonucleotide probes comprises a binding site for a second universal primer.

12. The method of claim 11, wherein the amplifying comprises PCR amplification of at least a portion of the oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules using the first universal primer and the second universal primer.

13. The method of any one of claims 1-12, wherein each of the plurality of oligonucleotide probes comprises a cellular label, a sample label, a location label, or any combination thereof.

14. The method of any one of claims 1-13, wherein the target specific region comprises 20 nt to 500 nt.

15. The method of any one of claims 1-14, wherein the sample comprises a single cell, a plurality of cells, a tissue sample, or any combination thereof.

16. The method of any one of claims 1-15, wherein the diverse set of unique molecular label sequences comprises at least 100 unique molecular label sequences.

17. The method of any one of claims 1-15, wherein the diverse set of unique molecular label sequences comprises at least 1,000 unique molecular label sequences.

18. The method of any one of claims 1-15, wherein the diverse set of unique molecular label sequences comprises at least 10,000 unique molecular label sequences.

19. The method of any one of claims 1-18, wherein at least 10 of the plurality of oligonucleotide probes comprise different target specific regions.

20. The method of any one of claims 1-18, wherein at least 100 of the plurality of oligonucleotide probes comprise different target specific regions.

21. The method of any one of claims 1-18, wherein at least 1,000 of the plurality of oligonucleotide probes comprise different target specific regions.

22. The method of any one of claims 1-21, wherein each of the plurality of RNA target molecules hybridizes to a single target specific region.

23. The method of any one of claims 1-21, wherein each of the plurality of RNA target molecules hybridizes to more than one different target specific regions.

24. The method of any one of claim 1-23, further comprising sequencing the plurality of amplicons.

25. The method of claim 24, wherein the sequencing comprises sequencing at least a portion of the molecular label sequence and at least a portion of the target specific region.

26. The method of claim 24 or 25, further comprising associating the sequence of the molecular label sequence with the sequence of the target specific region.

27. A kit for quantitative analysis of a plurality of nucleic acid target molecules in a sample comprising a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a target specific region, a molecular label sequence, a binding site for a first universal primer, and a binding site for a second universal primer, wherein the molecular label sequence is selected from a diverse set of unique molecular label sequences, and wherein the molecular label sequences of two of the plurality of oligonucleotide probes are different.

28. The kit of claim 27, further comprising a plurality of capture probes each comprising a second target specific region.

29. The kit of claim 28, wherein the plurality of capture probes is biotinylated.

30. The kit of claim 28 or 29, wherein the second target specific region comprises poly dT.

31. The kit of any one of claims 27-30, wherein each of the plurality of oligonucleotide probes comprises a cellular label, a sample label, a location label, or any combination thereof.

32. The kit of any one of claims 27-31, wherein the target specific region comprises 20 nt to 500 nt.

33. The kit of any one of claims 27-31, wherein the diverse set of unique molecular label sequences comprises at least 100 unique molecular label sequences.

34. The kit of any one of claims 27-31, wherein the diverse set of unique molecular label sequences comprises at least 1,000 unique molecular label sequences.

35. The kit of any one of claims 27-31, wherein the diverse set of unique molecular label sequences comprises at least 10,000 unique molecular label sequences.

36. The kit of any one of claims 27-35, wherein at least 10 of the plurality of oligonucleotide probes comprise different target specific regions.

37. The kit of any one of claims 27-35, wherein at least 100 of the plurality of oligonucleotide probes comprise different target specific regions.

38. The kit of any one of claims 27-35, wherein at least 1,000 of the plurality of oligonucleotide probes comprise different target specific regions.

39. The kit of any one of claims 27-38, wherein each of the plurality of oligonucleotide probes comprises a different molecular label sequence-target specific region combination.

40. The kit of any one of claims 27-39, comprising at least 1,000 oligonucleotide probes.

41. The kit of any one of claims 27-39, comprising at least 10,000 oligonucleotide probes.

42. The kit of any one of claims 27-39, comprising at least 100,000 oligonucleotide probes.

43. The kit of any one of claims 27-39, comprising at least 1,000,000 oligonucleotide probes.