Method and apparatus for flow-through hybridization

• US 6,638,760 B1
• Filed: 12/03/1998
• Issued: 10/28/2003
• Est. Priority Date: 11/25/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Fry, George A., Mehrpouyan, Majid, Lambert, Stephen M., Chen, Jer-Kang, Furniss, Vergine C., OʼNeill, Roger, Chiesa, Claudia
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/204,865
• Time in Patent Office: 1,790 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/93.5, 435/283.1, 435/287.1, 435/183, 435/287.2, 436/177, 436/807, 436/94, 536/24.3, 536/23.1, 536/24.33, 536/25.3
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention provides substrates and apparatuses for efficient, rapid and specific capture, and optimal recovery, of nucleic acids, as well as methods of their use. The substrate is porous in nature and has a capture polynucleotide capable of hybridizing to a target nucleic acid immobilized thereon. Upon flowing a sample containing or suspected of containing the target nucleic acid through the porous substrate, the target nucleic acid is rapidly captured. Following capture, the target nucleic acid can be efficiently recovered for subsequent use.

Claims

1. A flow-through device for capturing a target nucleic acid comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of polyethylene, polystyrene, polycarbonate and polypropylene and having immobilized thereon about 6×

10<sup>−

17</sup> to 6×

10<sup>−

16</sup> nmol/nm² of a capture polynucleotide which is capable of hybridizing to the target nucleic acid, and wherein said porous substrate is about 1 mm to 20 mm thick.

2. The flow-through device of claim 1 in which said porous substrate has an average pore size of about 1 μ

m to about 250 μ

m.

3. The flow-through device of claim 1 in which the porous substrate has a porosity in the range of about 25 to 80%.

4. The flow-through device of claim 1 or 6 in which the capture polynucleotide is covalently immobilized on the porous substrate via its 5′

- or 3′

-terminal residue.

5. The flow-through device of claim 4 further including a linker disposed between the porous substrate and the capture polynucleotide.

9. The flow-through device of claim 1 or 6 in which said capture polynucleotide is covalently attached to the porous substrate.

10. The flow-through device of claim 1 or 6 in which said capture polynucleotide is covalently attached to the porous substrate via a phosphodiester, phosphorothioate or phosphoramidate linkage.

11. The flow-through device of claim 1 or 6 in which said capture polynucleotide is covalently attached to the porous substrate via a carboxyamide linkage.

12. The flow-through device of claim 1 or 6 in which said capture polynucleotide is covalently attached to the porous substrate via a linker.

13. The flow-through device of claim 1 or 6 in which said porous substrate has a void volume in the range of about 1 μ

l/cm² to about 100 μ

l/cm².

14. The flow-through device according to claim 1 or 6 further comprising a housing in which the three-dimensional porous substrate is disposed.

15. The flow-through device of claim 14, in which said housing is selected from the group consisting of a syringe barrel, a pipette, a disposable pipette tip, a chromatography column, a spin column, a microchannel, a capillary and a multi-well plate.

16. A method of capturing a target nucleic acid from a sample, said method comprising flowing a sample containing or suspected of containing a target nucleic acid through a flow-through device according to claim 1 under conditions wherein said target nucleic acid and capture polynucleotide hybridize to one another to form a hybridized complex, thereby capturing the target nucleic acid.

18. The method of claim 16 or 17 in which said target nucleic acid is applied to the flow-through device under conditions wherein it hybridizes with said capture polynucleotide in less than one minute.

19. The method of claim 16 in which said porous substrate of said flow-through device has an average pore size of about 1 μ

m to about 250 μ

m.

21. The method of claim 16 or 17 in which said capture polynucleotide is covalently attached to the porous substrate of the flow-through device.

22. The method of claims 16 or 17 in which said capture polynucleotide is covalently attached to the porous substrate of the flow-through device via a phosphodiester, phosphorothioate or phosphoramidate linkage.

23. The method of claim 16 or 17 in which said capture polynucleotide is covalently attached to the porous substrate of the flow-through device via a carboxyamide linkage.

24. The method of claim 16 or 17 in which said capture polynucleotide is covalently attached to the porous substrate of the flow-through device via a linker.

25. The method of claim 16 or 17 in which said porous substrate of said flow-through device has a void volume in the range of 0.1 μ

l/cm² to about 100 μ

l/cm².

26. The method of claim 16 or 17 which further includes the step of washing said hybridized complex.

27. A method of determining whether a sample contains a target nucleic acid, said method comprising the steps of:

(a) flowing a sample suspected of containing a target nucleic acid through a flow-through device according to claim 1 or 6 under conditions wherein the target nucleic acid and capture polynucleotide hybridize; and

(b) detecting the presence of hybrids, wherein a positive detection indicates the presence of the target nucleic acid in the sample.

28. The method of claim 27, in which said target nucleic acid bears a reporter moiety and hybrids are detected by detecting the presence of said reporter moiety.

29. A kit for capturing a target nucleic acid of interest from a sample, comprising:

a) a flow-through device according to claim 1 or 6; and

b) a housing into which the flow-through device can be disposed.

30. A kit for capturing a target nucleic acid from a sample comprising:

a) a flow-through device according to claims 1 or 6; and

b) a capture polynucleotide capable of being covalently attached to the porous substrate.

31. The kit of claim 30 further including a linker capable of being covalently attached to the porous substrate and the capture polynucleotide.

32. The kit of claim 30 or 31 in which the porous substrate is activated with about 6×

10<sup>−

17</sup> to 9×

10¹⁵ nmol/nm² of a reactive group.

33. A kit for capturing a target nucleic acid from a sample comprising:

a) a flow-through device according to claims 1 or 6; and

b) means for generating a capture polynucleotide which is capable of hybridizing to the target nucleic acid and which is capable of being covalently attached to the porous substrate.

6. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of polyethylene, polystyrene, polycarbonate and polypropylene and having an average pore size of about 10 μ

m to about 100 μ

m and a porosity in the range of about 25 to 80% and having immobilized thereon a capture polynucleotide capable of hybridizing to the target nucleic acid.

7. The flow-through device of claim 6 in which said porous substrate is about 1 mm to 20 mm thick.

8. The flow-through device of claim 6 in which said porous substrate has immobilized thereon about 2×

10<sup>−

19</sup> to 2×

10<sup>−

15</sup> nmol/nm² of said capture polynucleotide.

17. A method of capturing a target nucleic acid from a sample, said method comprising flowing a sample containing or suspected of containing a target nucleic acid thorough a flow-through device according to claim 6 under conditions wherein said target nucleic acid and capture polynucleotide hybridize to one another to form a hybridized complex, thereby capturing the target nucleic acid.

20. The method of claim 17 in which the density or surface concentration of said capture polynucleotide is about 2×

10<sup>−

19</sup> to 2×

10<sup>−

15</sup> nmol/nm².

34. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of polyethylene, polystyrene, polycarbonate and polypropylene and having a porosity in the range of about 25 to 80% and having immobilized thereon a capture polynucleotide capable of hybridizing to the target nucleic acid.

40. The flow-through device of claim 34, 35, 36, 37, 38 or 39 in which said capture polynucleotide is covalently attached to the porous substrate.

41. The flow-through device of claim 34, 35, 36, 37, 38 or 39 in which said porous substrate has a void volume in the range of about 1 μ

l/cm² to about 100 μ

l/cm².

42. The flow-through device of claim 34, 35, 36, 37, 38 or 39 in which the capture polynucleotide is covalently immobilized on the porous substrate via its 5′

- or 3′

-terminal residue.

43. The flow-through device according to claim 34, 35, 36, 37, 38 or 39 further comprising a housing in which the three-dimensional porous substrate is disposed.

44. A method of capturing a target nucleic acid from a sample, said method comprising flowing a sample containing or suspected of containing a target nucleic acid through a flow-through device according to claim 34, 35, 36, 37, 38 or 39 under conditions wherein said target nucleic acid and capture polynucleotide hybridize to one another to form a hybridized complex, thereby capturing the target nucleic acid.

45. The method of claim 44 in which said target nucleic acid is applied to the flow-through device under conditions wherein it hybridizes with said capture polynucleotide in less than one minute.

35. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of glass, polyethylene, polystyrene, polycarbonate and polypropylene and having an average pore size of about 10 μ

m to about 100 μ

m and a porosity in the range of about 25 to 80% and having immobilized thereon about 6×

10<sup>−

17</sup> to 6×

10<sup>−

16</sup> nmol/nm² of a capture polynucleotide which is capable of hybridizing to the target nucleic acid.

36. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of glass, polyethylene, polystyrene, polycarbonate and polypropylene and having an average pore size of about 10 μ

m to about 100 μ

m and a porosity in the range of about 25 to 80% and having immobilized thereon a capture polynucleotide capable of hybridizing to the target nucleic acid, wherein said porous substrate is about 1 mm to 20 mm thick.

37. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of glass, polyethylene, polystyrene, polycarbonate and polypropylene and having an average pore size of about 10 μ

m to about 100 μ

m and having immobilized thereon about 6×

10<sup>−

17</sup> to 6×

10<sup>−

16</sup> nmol/nm² of a capture polynucleotide which is capable of hybridizing to the target nucleic acid, wherein said porous substrate is about 1 mm to 20 mm thick.

38. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of glass, polyethylene, polystyrene, polycarbonate and polypropylene and having a porosity in the range of about 25 to 80% and having immobilized thereon about 6×

10<sup>−

17</sup> to 6×

10−

16 nmol/nm² of a capture polynucleotide which is capable of hybridizing to the target nucleic acid, wherein said porous substrate is about 1 mm to 20 mm thick.

39. A flow-through device for capturing a target nucleic acid, comprising a three-dimensional porous substrate composed of a polymeric material selected from the group consisting of glass, polyethylene, polystyrene, polycarbonate and polypropylene and having an average pore size of about 1 μ

m to about 250 μ

m and a porosity in the range of about 25 to 80% and having immobilized thereon about 2×

10<sup>−

19</sup> to 2×

10<sup>−

15</sup> nmol/nm² of a capture polynucleotide which is capable of hybridizing to the target nucleic acid, wherein said porous substrate is about 1 mm to 20 mm thick.