Pesquisa nas coleções internacionais e nacionais de patentes
Algum conteúdo deste aplicativo está indisponível no momento.
Se esta situação persistir, por favor entre em contato conoscoFale conosco & Contato
1. (WO2019028166) HYDROGEL BEADS FOR NUCLEOTIDE SEQUENCING
Nota: O texto foi obtido por processos automáticos de reconhecimento ótico de caracteres.
Para fins jurídicos, favor utilizar a versão PDF.

WHAT IS CLAIMED IS:

1. A bead for performing nucleic acid reactions, comprising:

a hydrogel polymer; and

genetic material disposed within the hydrogel, wherein the bead comprises pores that allow diffusion of a reagent through the bead while retaining the genetic material.

2. The bead of Claim 1, wherein the bead has a diameter of about 2 μιη to about 120 μιη.

3. The bead of any one of Claims 1-2, wherein the hydrogel polymer comprises polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof.

4. The bead of Claim 3, wherein the hydrogel polymer comprises PEG-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), or PEG/PPO.

5. The bead of any one of Claims 1-4, wherein the genetic material is a cell, a nucleic acid, or a microbiome.

6. The bead of Claim 5, wherein the cell is a mammalian cell or bacterial cell.

7. The bead of any one of Claims 5-6, wherein the cell is an Escherichia coli cell, a Bacillus subtilis cell, an Aeromonas hydrophila cell, or a fibroblast cell.

8. The bead of Claim 5, wherein the nucleic acid is DNA or RNA of 300 base pairs or greater.

9. The bead of any one of Claims 1-8, wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs.

10. The bead of any one of Claims 1-9, wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase (Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (Tn5), primers (P5 and P7 adaptor sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations.

11. A method of encapsulating genetic material within a hydrogel bead, comprising: mixing genetic material with a hydrogel polymer in solution;

mixing the solution with an immiscible fluid to form a hydrogel bead encapsulating the genetic material, wherein the hydrogel bead comprises pores that allow diffusion of a reagent through the hydrogel bead while retaining the genetic material.

12. The method of Claim 11, wherein the hydrogel bead has a diameter of about 2 μιη to about 120 μιη.

13. The method any one of Claims 11-12, wherein the hydrogel polymer comprises polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof.

14. The method of Claim 13, wherein the hydrogel polymer comprises PEG-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), or PEG/PPO.

15. The method of any one of Claims 11-14, wherein the genetic material is a cell, a nucleic acid, or a microbiome.

16. The method of Claim 15, wherein the cell is a mammalian cell or bacterial cell.

17. The method of any one of Claims 15-16, wherein the cell is an Escherichia coli cell, a Bacillus subtilis cell, an Aeromonas hydrophila cell, or a fibroblast cell.

18. The method of Claim 15, wherein the nucleic acid is DNA or RNA of 300 base pairs or greater.

19. The method of any one of Claims 11-18, wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs.

20. The method of any one of Claims 11-19, wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase (Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (Tn5), primers (P5 and P7 adaptor sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations.

21. A method of encapsulating genetic material within a hydrogel bead, comprising: mixing an aqueous solution comprising a hydrogel polymer and genetic material with an immiscible fluid;

inputting the aqueous solution into a droplet generator; and

generating a hydrogel bead encapsulating the genetic material, wherein the hydrogel bead comprises pores that allow diffusion of a reagent through the hydrogel bead while retaining the genetic material.

22. The method of Claim 21, wherein the hydrogel bead has a diameter of about 2 μπι to about 150 μπι.

23. The method any one of Claims 21-22, wherein the hydrogel polymer comprises polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations or mixtures thereof.

24. The method of Claim 23, wherein the hydrogel polymer comprises PEG-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), or PEG/PPO.

25. The method of any one of Claims 21-24, wherein the genetic material is a cell, a nucleic acid, or a microbiome.

26. The method of Claim 25, wherein the cell is a mammalian cell or bacterial cell.

27. The method of any one of Claims 25-26, wherein the cell is an Escherichia coli cell, a Bacillus subtilis cell, an Aeromonas hydrophila cell, or a fibroblast cell.

28. The method of Claim 25, wherein the nucleic acid is DNA or RNA of 300 base pairs or greater.

29. The method of any one of Claims 21-28, wherein the reagent comprises enzymes, chemicals, and primers having a size of less than 50 base pairs.

30. The method of any one of Claims 21-29, wherein the reagent comprises lysozyme, proteinase K, random hexamers, polymerase (Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (Tn5), primers (P5 and P7 adaptor sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations.

31. A method of preparing a nucleic acid library from genetic material encapsulated within a bead, comprising:

obtaining a bead of any one of Claims 1-10;

amplifying genetic material encapsulated within the bead;

performing a tagmentation reaction on the genetic material encapsulated within the bead; and

sequencing the genetic material, thereby generating a nucleic acid library encapsulated within the bead.

32. The method of Claim 31, wherein the genetic material is a cell, and further comprising lysing the cell and extracting nucleic acids.

33. The method of Claim 32, wherein the cell is lysed with lysozyme and treated with proteinase K to extract nucleic acids.

34. The method of any one of Claims 31-33, wherein the tagmentation reaction comprises contacting genetic material with a transposase mixture comprising adapter sequences and transposomes.

35. The method of any one of Claims 31-34, further comprising seeding the nucleic acid library on a solid support.

36. The method of Claim 35, wherein seeding comprises degrading the bead to release the nucleic acid library from the bead.

37. The method of Claim 36, wherein the bead is degraded by contacting the bead with a cleavage mix or by heating the bead to about 90°C to release the nucleic acid library.

38. The method of Claim 37, wherein the cleavage mix comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP ), or tris(3 -hydroxy propyl)phosphine (THP).

39. The method of any one of Claims 35-38, wherein the solid support is a flow cell device.