Method For Parallelly Sequencing A Nucleic Acid Mixture By Using a Continuous Flow System

Abstract

The invention relates to a method for the parallel sequencing of nucleic acids, comprising the steps: 1. Providing a porous support possessing areas distinguished by immobilized nucleic acid molecules,2. Inserting the support of step (1) into a flow through arrangement,3. simultaneously determining at least a part of the nucleotide sequence of at least a part of the nucleic acid molecules.

Description

The invention is described in more detail by the drawings, showing the following:

FIG. 1 a porous support for carrying out the method of the invention;

FIG. 2 the immobilization of nucleic acids within the porous support's channels;

FIG. 3 the amplification of an appropriately diluted solution of nucleic acid molecules in the porous support s channels;

FIG. 4 a flow through arrangement for carrying out the method of the invention;

FIG. 5 a possible function structure for the flow through arrangement of FIG. 4;

FIG. 6 the sequencing of immobilized nucleic acid molecules by means of reversible chain terminating nucleotides;

FIG. 7 the simultaneous sequencing of the immobilized nucleic acid molecules in several different areas of the porous support.

FIG. 1 indicates a porous support for carrying out the method of the invention, with

• • 1 the porous support's top side,
  • 2 the porous support's bottom side,
  • 3 channels running through from the porous support's top side to its bottom side,
  • 4 a partition between two adjoining channels.

FIG. 2 shows the immobilization of nucleic acids within the porous support's channels, with

• • 1 a device for transferring the nucleic acid solutions to the porous support, such as, e.g., a pin, a capillary, or a jet,
  • 2 a desired volume of a solution of nucleic acid molecules of the same sort, which due to the action of capillary forces is absorbed, after transfer to the porous support's top side, by the support's channels,
  • 3 a detail of the porous support,
  • 4 a solution of nucleic acid molecules absorbed, by means of capillary forces, by the porous support's channels,
  • 5 filled channels,
  • 6 non-filled channels,
  • 7 dissolved nucleic acid molecules of the same sort,
  • 8 an atom group mediating the terminal irreversible immobilization of a nucleic acid molecule (one partner of a specific binding pair),
  • 9 an atom group capable of specifically binding to the atom group (8) (the other partner of the same specific binding pair),
  • 10 a channel's wall,
  • 11 nucleic acid molecules of the same sort, immobilized to the channel's wall.

FIG. 3 depicts the amplification of an appropriately diluted solution of nucleic acid molecules in the porous support's channels, with

• • 1 the porous support,
  • 2 the filling of essentially all the support's channels by a diluted solution of different nucleic acid molecules in an amplification mixture, containing the reagents required for carrying out an amplification reaction,
  • 3 channels filled by the solution of (2),
  • 4 amplification of single molecules in those channels having contained, after filling, one amplifiable nucleic acid molecule each, to numerous copies of these molecules,
  • 5 channels in which an amplification of one nucleic acid molecule to numerous copies has taken place,
  • 6 channels in which no amplification of nucleic acid molecules has taken place.

FIG. 4 indicates a flow through arrangement for carrying out the method of the invention, with FIG. 4a: flow through arrangement after inserting the porous support and FIG. 4b: flow through arrangement in operation, with

• • 1 holding device of the support,
  • 2 O-seal,
  • 3 porous support,
  • 4 current of an appropriately tempered reagent solution for sequencing of the nucleic acid molecules immobilized to the porous support,
  • 5 lid,
  • 6 observation window,
  • 7 detector.

FIG. 5 shows a possible function structure of the flow through arrangement of FIG. 4.

FIG. 6 shows the sequencing of immobilized nucleic acid molecules by means of reversible chain terminating nucleotides, with

• • 1 a channel's wall,
  • 2 a nucleic acid molecule having a terminal hairpin structure,
  • 3 a C nucleotide, reversibly protected at its 3′position, by a fluorescently labeled protecting group, against further strand extension,
  • 4 the C nucleotide of (3) after cleaving off the protecting group, upon restoration of a 3′OH group,
  • 5 an A nucleotide, reversibly protected at its 3′end, by a fluorescently labeled protecting group, against further strand extension,
  • 6 a strand extension by one base, made possible by the incorporation of a fluorescently labeled dCTP derivative, reversibly protected at its 3′position,
  • 7 the detection, with identification of the nucleotide incorporated last, of the fluorescent label incorporated into the strand in step (6), followed by cleaving off the fluorescent protecting group,
  • 8 a further strand extension by one base, enabled by incorporation of a fluorescently labeled dATP derivative reversibly protected at its 3′position,
  • 9 repetition of the steps detection, cleaving off, and strand extension by one base, until the desired read length is achieved.

FIG. 7 shows the simultaneous sequencing of nucleic acid molecules immobilized to several different areas of the porous support, with

• • 1 a detail of the support containing different areas, the signals obtained from the sequencing of a first base being identified by different filling patterns in the figure,
  • 2 a detail of the support containing the same areas, the signals obtained from the sequencing of a second base being identified by different filling patterns in the figure,
  • 3 a detail of the support containing the same areas, the signals obtained from the sequencing of an n^th base being identified by different filling patterns in the figure,
  • 4 two different areas of the porous support, each comprising one or more channels,
  • 5 a superimposition of the results obtained, upon sequencing the first base up to the n^th base, for all covered areas,
  • 6 the sequencing results obtained in (5) for the first to the n^th base of the nucleic acid molecules immobilized to the covered areas.

Claims

1.-31. (canceled)

32. A method for the parallel sequencing of nucleic acids, comprising the steps: (1) providing a porous support possessing areas distinguished by immobilized nucleic acid molecules;(2) inserting the support of step (1) into a flow through arrangement; and(3) simultaneously determining at least a part of the nucleotide sequence of at least part of the nucleic acid molecules.

33. The method of claim 32, wherein the porous support is formed to have two parallel even surfaces and has a top side and a bottom side.

34. The method of claim 33, wherein the porous support possesses channels which are essentially parallel to each other and via which the top side and the bottom side communicate with each other.

35. The method of claim 34, wherein the channels' diameter is between 0.5 μm and 50 μm.

36. The method of claim 35, wherein the channels' diameter is between 1 μm and 25 μm.

37. The method of claim 32, wherein the porous support is made from glass.

38. The method of claim 37, wherein the porous support is a glass capillary array.

39. The method of claim 32, wherein the porous support is made from silicon.

40. The method of claim 32, wherein the nucleic acid molecules being positioned in the areas of step (1) have been transferred to the support as preformed nucleic acid solutions.

41. The method of claim 40, wherein the transfer has been made by pins, capillaries, or ink jet technology.

42. The method of claim 32, wherein the nucleic acid molecules being positioned in the areas of step (1) have been generated by amplification within the support's hollow spaces.

43. The method of claim 42, wherein at the beginning of the amplification, there are on average at most 0.5 amplifiable nucleic acid molecules within a hollow space.

44. The method of claim 43, wherein at the beginning of the amplification, there are on average at most 0.2 amplifiable nucleic acid molecules within a hollow space.

45. The method of claim 43, wherein at the beginning of the amplification, there are on average between 0.1 and 0.02 amplifiable nucleic acid molecules within a hollow space.

46. The method of claim 42, wherein upon amplification at least 106 copies of a starting molecule are generated.

47. The method of claim 46, wherein upon amplification at least 107 copies of a starting molecule are generated.

48. The method of claim 47, wherein upon amplification at least 108 copies of a starting molecule are generated.

49. The method of claim 32, wherein the sequencing of the nucleic acid molecules is carried out by incorporation of nucleotide triphosphates and by determination of reaction side products.

50. The method of claim 32, wherein the sequencing of the nucleic acid molecules is carried out by incorporation of labeled nucleotides.

51. The method of claim 32, wherein the sequencing of the nucleic acid molecules is carried out by incorporation of reversibly labeled nucleotides.

52. The method of claim 32, wherein the sequencing of the nucleic acid molecules is carried out by incorporation of labeled reversible chain terminating nucleotides.

53. The method of claim 32, wherein the support has at least 103 areas.

54. The method of claim 53, wherein the support has at least 104 areas.

55. The method of claim 54, wherein the support has at least 105 areas.

56. The method of claim 55, wherein the support has at least 106 areas.

57. The porous support of claim 33, wherein the walls have a coating appropriate for the immobilization of nucleic acid molecules.

58. The porous support of claim 53, wherein the walls have a coating appropriate for the immobilization of nucleic acid molecules.

59. The porous support of claim 33, comprising areas where in each case a plurality of essentially identical nucleic acid molecules is positioned.

60. The porous support of claim 53, comprising areas where in each case a plurality of essentially identical nucleic acid molecules is positioned.

61. The porous support of claim 33, comprising areas where in each case a plurality of essentially identical nucleic acid molecules is positioned, the areas at least in some cases consisting of a single hollow space each.

62. The porous support of claim 53, comprising areas where in each case a plurality of essentially identical nucleic acid molecules is positioned, the areas at least in some cases consisting of a single hollow space each.

63. A flow through arrangement comprising: (a) at least two spaces connected by a porous support;(b) a means for establishing a pressure difference;(c) a detector;(d) where appropriate, a source of radiation appropriate to fluorescence excitation, and(e) where appropriate, containers for the storage of reagents for performing amplification reactions and/or sequencing reactions.

64. A method for the massively parallel sequencing of nucleic acids, comprising the steps: (1) providing porous support;(2) introducing to the porous support's hollow spaces an amplification mixture, containing amplifiable nucleic acid molecules;(3) performing an amplification of the nucleic acid molecules in the support's hollow spaces;(4) contacting the porous support with a surface, this step optionally having been carried out before performing the amplification;(5) immobilization to the surface of at least a portion of the amplified nucleic acid molecules of step (3); and(6) simultaneous determination of at least a portion of the nucleotide sequence of at least a portion of the nucleic acid molecules immobilized to the surface.

65. The method as claimed in claim 32, wherein after immobilization the porous support is removed from the surface.