Capturing and amplifying polynucleotides using molecules and particles

Patent metadata

Field	Value
Publication number	US-2025188529-A1
Application number	US-202418977460-A
Country	US
Kind code	A1
Filing date	Dec 11, 2024
Priority date	Dec 12, 2023
Publication date	Jun 12, 2025
Grant date	—

Abstract

In some examples, a device includes a flowcell including wells, and a plurality of molecules. Each molecule may include a single respective polynucleotide. At least some of the wells are coupled to a single respective one of the molecules such that a single respective polynucleotide is coupled to those wells. The device also may include a plurality of particles. Each particle may include amplification primers and may be coupled to a single one of the wells via hybridization between an amplification primer of that particle and the polynucleotide of the molecule coupled to that well.

First claim

What is claimed is:

1 . A device, comprising:
a flowcell comprising wells; a plurality of molecules, each molecule comprising a single respective polynucleotide, wherein at least some of the wells are coupled to a single respective one of the molecules such that a single respective polynucleotide is coupled to those wells; and a plurality of particles, each particle comprising amplification primers and being coupled to a single one of the wells via hybridization between an amplification primer of that particle and the polynucleotide of the molecule coupled to that well.

2 . The device of claim 1 , wherein each of the molecules comprises:
a dendritic core to which the single respective polynucleotide is coupled; and a plurality of dendrons, each of the dendrons comprising an inert, elongated polymer comprising a first end coupled to the dendritic core and a second end.

3 . The device of claim 2 , wherein the dendrons and the dendritic core are disposed within a corresponding one of the wells.

4 . The device of claim 2 or claim 3 , wherein the dendrons and the dendritic core substantially fill a corresponding one of the wells.

5 . The device of any one of claims 2 to 4 , wherein the dendrons are covalently bonded to the corresponding one of the wells.

6 . The device of any one of claims 2 to 5 , wherein the polynucleotide is covalently bonded to the dendritic core.

7 . The device of any one of claims 1 to 6 , wherein the wells are covalently bonded to the molecules.

8 . The device of any one of claims 1 to 4 or 6 , wherein the molecules are held within the wells using a non-covalent force.

9 . The device of claim 1 or claim 8 , wherein each of the molecules comprises a protein to which the single respective polynucleotide is coupled.

10 . The device of claim 9 , wherein the protein comprises an antibody.

11 . The device of claim 10 , wherein the single respective polynucleotide is coupled to an antigen for which the antibody is selective.

12 . The device of any one of claims 1 to 11 , wherein the polynucleotide extends outside of the respective well.

13 . The device of any one of claims 1 to 12 , wherein the polynucleotide is single-stranded.

14 . The device of any one of claims 1 to 13 , wherein each of the molecules has a hydrodynamic diameter which is about 60% to about 100% of a diameter of the respective well.

15 . The device of any one of claims 1 to 14 , wherein the wells have a diameter between about 10 nm and about 200 nm.

16 . The device of any one of claims 1 to 15 , wherein the molecules have a diameter between about 10 nm and about 200 nm.

17 . The device of any one of claims 1 to 16 , wherein a pitch of the wells is at least five times a length of the single-stranded polynucleotides.

18 . The device of any one of claims 1 to 17 , each particle further comprising a hydrogel to which the plurality of amplification primers is coupled.

19 . The device of any one of claims 1 to 18 , wherein the polynucleotide comprises an amplification adapter that is hybridized to the one of the amplification primers.

20 . A method of amplifying polynucleotides, the method comprising:
flowing a plurality of molecules into a flowcell comprising wells, each molecule comprising a single respective polynucleotide; coupling at least some of the wells to a single respective one of the molecules such that a single respective polynucleotide is coupled to those wells; flowing a plurality of particles into the flowcell, each particle comprising amplification primers; at each well, hybridizing an amplification primer of one of the particles to the respective polynucleotide which is coupled to that well; extending that amplification primer to generate a first amplicon of the respective polynucleotide which is coupled to that well; and using the amplification primers of that particle to generate amplicons of the first amplicon.

21 . The method of claim 20 , wherein each of the molecules comprises:
a dendritic core to which the single respective polynucleotide is coupled; and a plurality of dendrons, each of the dendrons comprising an inert, elongated polymer comprising a first end coupled to the dendritic core and a second end.

22 . The method of claim 21 , comprising disposing the dendrons and the dendritic core within a corresponding one of the wells.

23 . The method of claim 21 or claim 22 , wherein the dendrons and the dendritic core substantially fill a corresponding one of the wells.

24 . The method of any one of claims 21 to 23 , further comprising covalently bonding the dendrons to the corresponding one of the wells.

25 . The method of any one of claims 21 to 24 , further comprising covalently bonding the polynucleotide to the dendritic core.

26 . The method of claim 25 , wherein covalently bonding the polynucleotide to the dendritic core comprises:
contacting a precursor of the dendritic molecule with a template polynucleotide, wherein the precursor of the dendritic molecule comprises a capture primer covalently coupled to the dendritic core, and wherein the template polynucleotide comprises an adapter; hybridizing the adapter to the capture primer; extending the capture primer using the template polynucleotide to form a duplex; and dehybridizing the template polynucleotide from the duplex to leave the polynucleotide covalently coupled to the dendritic core.

27 . The method of any one of claims 20 to 26 , comprising covalently bonding the wells to the molecules.

28 . The method of any one of claims 21 to 23, 25, or 26 , wherein the molecules are held within the wells using a non-covalent force.

29 . The method of claim 21 or claim 28 , wherein each of the molecules comprises a protein to which the single respective polynucleotide is coupled.

30 . The method of claim 29 , wherein the protein comprises an antibody.

31 . The method of claim 30 , wherein the single respective polynucleotide is coupled to an antigen for which the antibody is selective.

32 . The method of any one of claims 20 to 31 , wherein the polynucleotide extends outside of the respective well.

33 . The method of any one of claims 20 to 32 , wherein the polynucleotide is single-stranded.

34 . The method of any one of claims 20 to 33 , wherein each of the molecules has a hydrodynamic diameter which is about 60% to about 100% of a diameter of the respective well.

35 . The method of any one of claims 20 to 34 , wherein the wells have a diameter between about 10 nm and about 200 nm.

36 . The method of any one of claims 20 to 35 , wherein the molecules have a diameter between about 10 nm and about 200 nm.

37 . The method of any one of claims 20 to 36 , wherein a pitch of the wells is at least five times a length of the single-stranded polynucleotides.

38 . The method of any one of claims 20 to 37 , each particle further comprising a hydrogel to which the plurality of amplification primers is coupled.

39 . The method of any one of claims 20 to 38 , wherein the polynucleotide comprises an amplification adapter that hybridizes to the one of the amplification primers.

Assignees

• Illumina Inc

Inventors

• Von Hatten Xavier
• Artioli Gianluca
• Nguyen Nam
• Boutell Jonathan

Classifications

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• B01L2300/0829

  Multi-well plates; Microtitration plates · CPC title

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• B01L3/502

  with fluid transport, e.g. in multi-compartment structures · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title