Methods and systems of nucleic acid sequencing

• US 20030219770A1
• Filed: 11/08/2002
• Published: 11/27/2003
• Est. Priority Date: 11/08/2001
• Status: Abandoned Application

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Murphy, Kathleen M., Eshleman, James R.
• Application Number:
  US10/291,205
• Publication Number:
  US 20030219770A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• 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 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

Methods for the simultaneous sequencing of multiple nucleic acid molecules are provided. Preferred methods include simultaneous single-direction sequencing of multiple genes or forward and reverse sequencing from a single gene, within a single reaction vessel. Additional methods of the invention include combined amplification and sequencing of nucleic acids, from a variety of sources, within a single reaction and wherein nucleic acid products also can be simultaneously analyzed, and where the reaction can be either bidirectional or long unidirectional. Additional methods encompass combined amplification and sequencing of multiple nucleic acid molecules simultaneously.

Claims

1. A method for substantially simultaneously sequencing multiple nucleic acid targets, comprising:

providing a plurality of nucleic acid targets;

providing a plurality of primers;

annealing of the primers to target sequences of the nucleic acid targets;

sequencing the nucleic acid targets using the primers to obtain a pool of sequence data; and

,

analyzing the sequence data without the need to separate the pool of sequence data prior to analysis.

2. The method of claim 1 wherein the pool of sequence data is analyzed substantially simultaneously within a single lane or capillary.

3. The method of claim 1 or 2 wherein the nucleic acid targets are DNA or RNA molecules.

4. The method of claim 3 wherein the nucleic acid targets are single stranded DNA molecules.

5. The method of claim 3 wherein the nucleic acid targets are double stranded DNA molecules.

6. The method of any one of claims 1 through 5 wherein the nucleic acid targets are cDNA, genes or fragments thereof, or non-coding DNA.

7. The method of claim 6 wherein the nucleic acid targets are from the same gene or fragments thereof.

8. The method of claim 6 wherein the DNA nucleic acid targets are from different genes or fragments thereof.

9. The method of any one of claims 1 through 8 wherein the primers are of varying lengths, modifications, and/or size.

10. The method of any one of claims 1 through 9 wherein the primers are modified to comprise abasic regions.

11. The method of any one of claims 1 through 10 wherein the primers are comprised of non-template or template 5′

tails of varying lengths and/or compositions of nucleotides or other molecules.

12. The method of any one of claims 1 through 11 wherein the primers are specific for different target DNA sequences.

13. The method of any one of claims 1 through 11 wherein the primers are specific for the same target DNA sequences.

14. The method of any one of claims 1 through 11 wherein the desired length of the sequence data is varied according to the design of the primer used.

15. The method of claim 14 wherein the shortest desired length of sequence data is at least about one nucleic acid base.

16. The method of any one of claims 1 through 15 wherein the sequencing reaction is uni-directional.

17. The method of any one of claims 1 through 15 wherein the sequencing reaction is bi-directional.

18. The method of any one of claims 1 through 17 wherein the sequencing reaction does not require the separation of the nucleic acids to be separated into different reaction vessels.

19. The method of any one of claims 1 through 18 wherein the nucleic acid targets are pooled from a variety of sources.

20. The method of any one of claims 1 through 19 wherein the method steps are performed in a single reaction vessel.

21. The method of any one of claims 1 through 20 wherein each step of the method is performed in a single reaction vessel.

22. The method of any one of claims 1 through 21 wherein the sequencing reaction of multiple DNA oligonucleotides, or fragments thereof, is performed in a single step without the need to separate each oligonucleotide into separate reaction vessels.

23. The method of any one of claims 1 through 12 wherein the sequence data are analyzed without the need to separate each sequence obtained from said sequencing reaction, before analysis of said data.

24. The method of any one of claims 1 through 23 wherein the plurality of target nucleic acid molecules are amplified by polymerase chain reactions, prior to sequencing.

25. The method of claim 24 wherein the polymerase chain reaction primers are removed from the amplified products prior to sequencing.

26. The method of claim 24 wherein the polymerase chain reaction primers are removed by enzymatic or physical treatment.

27. The method of claim 24 wherein the reverse polymerase chain reaction primers are functionally removed using uracil N-DNA-glycosylase.

40. The method of any one of claims 24, 25 or 35 through 39 wherein the polymerase chain reaction is a standard polymerase chain reaction, a ligase chain reaction, reverse transcriptase polymerase chain reaction, Rolling Circle polymerase chain reaction, multiplex polymerase chain reaction, isothermal amplification, strand displacement, and the like.

67. The method of any one of claims 1 through 66 wherein the primers are modified by conjugate molecules to further increase the binding affinity and hybridization rate of these oligonucleotides to a target.

68. The method of claim 67 wherein the conjugate molecules are selected from the group consisting of cationic amines, intercalating dyes, antibiotics, proteins, peptide fragments, and metal ion complexes.

69. The method of any one of claims 1 through 68 wherein the primers are modified to increase avidity of binding and/or hybridization rates between a primer and its target nucleic acid.

70. The method of claim 69 wherein the primers are comprised of 2′

modifications to a ribofuranosyl ring of a primer or any other modification.

71. The method of claim 70 wherein said modification comprises a 2′

-O-methyl substitution.

72. The method of any one of claims 1 through 71 wherein one or more of the primers are modified to produce varying lengths of amplified and/or sequenced product.

73. The method of any one of claims 1 through 72 wherein one or more of the primers are modified by capping or blocking 3′

ends of primers to prevent or inhibit their use as templates for nucleic acid polymerase activity.

74. The method of claim 73 wherein the primers are capped by addition of 3′

deoxyribonucleotides or 3′

, 2′

-dideoxynucleotide residues.

75. The method of claim 73 wherein one or more of the primers are capped using non-nucleotide linkers or non-complementary nucleotide residues at the 3′

terminus.

76. The method of claim 75 wherein one or more of the primers have alkane-diol modifications.

77. A method of any one of claims 1 through 76 wherein a disease or disorder is identified.

78. A kit comprising components for performing any of the methods of claims 1 through 77.

84. The kit of any one of claims 77 through 83 wherein the kit comprises a forward or reverse primer that comprises an abasic region.

85. The kit of any one of claims 77 through 84 wherein the kit comprises modified reverse primer that comprise non-template nucleic acids and is longer in length in relation to the forward primer.

86. The kit of claim 85 wherein the one or more modified reverse primers comprise a polythymidine, polycytosine, polyguanine, polyadenine, polyuracil, polyinosine, or other nucleic acid or non-nucleic acid containing tail.

87. The kit of any one of claims 77 through 86 wherein the kit comprises a modified forward primer that comprise non-template nucleic acids and is longer in length in relation to the reverse primer.

88. The kit of any one of claims 77 through 87 wherein the one or more modified forward primers comprise a polythymidine, polycytosine, polyguanine, polyadenine, polyuracil, polyinosine, or other nucleic acid or non-nucleic acid containing tail.

89. Use of a kit of any one of claims 77 through 88 in a diagnostic assay.

90. Use of a method of any one of claims 1 through 89 on a microarray platform.

91. Use of a kit of any one of claims 77 through 88 wherein the primers are used at unequal molar ratios to perform combined amplification and sequencing.

92. Use of a method of any one of claims 1 through 70, wherein the dideoxynucleoside triphosphate is a fluorescently labelled dye terminator or where the primer is fluorescently labelled in the presence of unlabelled ddNTPs.

93. Use of a method of claim 92 wherein the dNTPs or ddNTPs are replaced by their ribonucleotide counterparts.

94. Use of a kit of any one of claims 77 through 88 wherein the dideoxynucleoside triphosphate is a fluorescently labelled dye terminator or where the primer is fluorescently labelled in the presence of unlabelled ddNTPs.

95. Use of a kit of claim 94 wherein the dNTPs or ddNTPs are replaced by their ribonucleotide counterparts

28. A method for simultaneously amplifying and sequencing a single nucleic acid molecule or a plurality of nucleic acid molecules, comprising:

providing a single or plurality of target nucleic acid molecules, and a single or a plurality of forward and reverse nucleic acid primer molecules, wherein each primer molecule hybridizes to a distinct area of the target nucleic acid molecules;

amplifying said target nucleic acid molecules;

wherein,

deoxyribonucleosides triphosphates are present during the amplifying;

wherein,

the number of amplifying cycles are determined by the added concentration of deoxyribonucleosides triphosphates;

wherein,

as the amplifying cycles consume the added deoxyribonucleosides triphosphates, the concentrations of free deoxyribonucleosides triphosphates decrease thereby raising the relative concentration of di-deoxyribonucleoside triphosphates.

29. The method of claim 28 wherein deoxyribonucleases triphosphates are added in admixture with the nucleic acid molecules prior to the amplifying.

30. The method of claim 28 or 29 wherein the method steps are performed in a single reaction vessel.

31. The method of claim 28 or 29 wherein each step of the method is performed in a single reaction vessel.

32. The method of any one of claims 28 through 31 wherein a single reaction is provided that comprises the plurality of target nucleic acid molecules and the plurality of forward and reverse nucleic acid primer molecules.

33. The method of any one of claims 28 through 32 wherein varying concentration of deoxyribonucleosides triphosphates are added prior to amplification and the number of amplifying cycles are determined, at least in part, by the added concentration of deoxyribonucleosides triphosphates.

34. The method of any one of claims 28 through 33 wherein a sequencing reaction is favored over amplification as the concentration of di-deoxyribonucleoside triphosphates increase relative to free deoxyribonucleoside triphosphates.

35. The method of any one of claims 28 through 34 wherein the amplifying reaction comprises a polymerase chain reaction.

36. The method of any one of claims 28 through 35 wherein amplification of target nucleic acid molecules via polymerase chain reaction and sequencing of polymerase chain reaction products is performed in a single reaction vessel without the need to process or clean-up the amplified products prior to the sequencing reaction.

37. The method of any one of claims 28 through 36 wherein the concentration of added free deoxyribonucleosides triphosphates determines the number of amplification cycles.

38. The method of any one of claims 28 through 37 wherein the concentration of di-deoxyribonucleosides triphosphates relative to the deoxyribonucleosides triphosphates increases as the deoxyribonucleosides triphosphates are consumed during amplification cycles.

39. The method of any one of claims 28 through 38 wherein the relative free concentrations deoxyribonucleosides triphosphates to di-deoxyribonucleosides triphosphates favors a shift from the amplification reaction to a sequencing reaction.

41. The method of any one of claims 28 through 40 wherein the target nucleic acid molecules are DNA or RNA, and the like.

42. The method of any one of claims 28 through 41 wherein the target nucleic acid molecules are single stranded.

43. The method of any one of claims 28 through 42 wherein the target nucleic acid molecules are double stranded.

44. The method of any one of claims 41 through 43 wherein the target nucleic acid molecules are comprised of eDNA, or genes or fragments thereof, or non-coding nucleic acids or fragments thereof.

45. The method of any one of claims 41 through 44 wherein the target nucleic acid molecules are from the same gene or fragments thereof.

46. The method of any one of claims 41 through 44 wherein the target nucleic acid molecules are from different genes or fragments thereof.

47. The method of any one of claims 28 through 46 wherein the plurality of forward and reverse nucleic acid primer molecules each hybridizes to a distinct area of the target nucleic acid molecules and said primers are of varying lengths, modifications, and sizes.

48. The method of any one of claims 28 through 47 wherein the primers are present at non-equal molar ratios.

49. The method of claim 48 wherein said primers are unmodified, modified, or a combination thereof.

50. The method of any one of claims 28 through 49 wherein one or more of the primers are modified to comprise abasic regions.

51. The method of any one of claims 28 through 50 wherein one or more of the primers comprise non-template or templated 5′

tails of varying lengths.

52. The method of any one of claims 28 through 51 wherein the primers are specific for different target nucleic acid sequences.

53. The method of any one of claims 28 through 51, wherein the primers are specific for the same target nucleic acid sequences.

54. The method of any one of claims 28 through 53 wherein the forward or reverse primer is targeted to a different or same position on the amplified product.

55. The method of claim 54 wherein the modified forward or reverse primers comprises an abasic region.

56. The method of claim 55 wherein the modified reverse primer comprises non-template nucleic acids such as polythymidine tails and is longer in length in relation to the forward primer.

57. The method of claim 55 wherein the modified forward primer comprises non-template nucleic acids such as polythymidine tails and is longer in length in relation to the reverse primer.

58. The method of any one of claims 28 through 57 wherein the forward and reverse primers produce amplified products of varying lengths.

59. The method of any one of claims 28 through 58 wherein the sequencing reaction is uni-directional.

60. The method of any one of claims 28 through 58 wherein the sequencing reaction is bi-directional.

61. The method of any one of claims 28 through 60 wherein the amplification and sequencing reactions do not require the separation of the nucleic acids into different reaction vessels.

62. The method of any one of claims 28 through 60 wherein the amplification and sequencing reactions are performed in a single step.

63. The method of any one of claims 28 through 62 wherein sequencing data obtained from the sequencing reaction is analyzed in a single well on a gel or capillary.

64. The method of claim 63 wherein the sequencing data is analyzed by immobilizing the reverse primer on a solid support.

65. The method of claim 63 wherein the sequencing data is analyzed by using a modified reverse primer such that its migration in the gel or column is slower relative to any other product produced during the amplification and sequencing reactions.

66. The method of claim 65 wherein the reverse primer is modified by biotinylation, blocking group, use of branched primers and the like.

79. A kit suitable for substantially simultaneously sequencing multiple oligonucleotides, pooled from a variety of sources, in a single reaction using a single reaction vessel, the kit comprising a plurality of modified primers and a plurality of oligonucleotides.

80. A kit suitable for amplifying and substantially simultaneously sequencing a single nucleic acid molecule or a plurality of nucleic acid molecules in a single reaction within a single reaction vessel, the kit comprising a single or a plurality of target nucleic acid molecule(s), and a single or plurality of forward and reverse nucleic acid primer molecule(s), and reagents for an amplification reaction.

81. The kit of claim 80 comprising deoxyribonucleosides triphosphates.

82. The kit of claim 80 or 81 wherein the one or more of the primers are modified.

83. The kit of any one of claims 80 through 82 wherein the forward primer is targeted to a different position on the amplified product and the reverse primer is of longer length and modified.