Mass label linked hybridisation probes

• US 20050042625A1
• Filed: 12/30/2003
• Published: 02/24/2005
• Est. Priority Date: 01/15/1997
• Status: Abandoned Application

• Current Assignee: Xzillion GmbH & Company
• Original Assignee: Xzillion GmbH & Company
• Inventors: Johnstone, Robert Alexander Walker, Schmidt, Gunter, Thompson, Andrew Hugin
• Application Number:
  US10/747,229
• Publication Number:
  US 20050042625A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

An array of hybridization probes, each of which comprises a mass label linked to a known base sequence of predetermined length, wherein each mass label of the array, optionally together with the known base sequence, is relatable to that base sequence by mass spectrometry.

Claims

1-50. -50. (Canceled)

51. An array of hybridisation probes, each of which comprises a mass label linked to a known base sequence of predetermined length, wherein each mass label of the array, optionally together with the known base sequence, is relatable to that base sequence by mass spectometry, and wherein each mass label comprises a photo-excitation group.

52. The array according to claim 51, wherein each mass label is uniquely identifiable in relation to every other mass label in the array.

53. The array according to claim 51, wherein the predetermined length of the base sequence is from 2 to 25.

54. The array according to any claim 51, wherein each mass label is cleavably linked to its respective known base sequence and is relatable to its base sequence by mass spectrometry when released therefrom.

55. The array according to claim 54, wherein each mass label is cleavably linked to the known base sequence by a collision-cleavable, photo-cleavable, chemically-cleavable or thermally-cleavable link.

56. The array according to claim 54, wherein each mass label is cleavably linked to the known base sequence by a link which cleaves when in a mass spectrometer.

57. The array according to claim 54, wherein each mass label is negatively-charged under ionisation conditions.

58. The array according to claim 51, wherein the known base sequence comprises a sticky end of an adaptor oligonucleotide containing a recognition site for a restriction endonuclease which cuts at a predetermined displacement from the recognition site.

59. The array according to claim 51, wherein the known base sequence has linked thereto a plurality of identical mass labels.

60. The array according to claim 51, wherein the photo-excitation group is an excitable ionisation agent and is suitable for performing matrix-assisted laser desorption ionisation.

61. The array according to claim 60, wherein the photo-excitation group is selected from nicotinic acid, sinapinic acid or cinnamic acid.

82. A method for reading an oligonucleotide chip using an array as defined in claim 51.

83. A method for identifying an oligonucleotide binding agent in a competitive binding assay using an array as defined in claim 51.

84. A method to probe for predetermined sequences in a polymerase chain reaction or a ligase chain reaction using an array as defined in claim 51.

85. A method for determining hybridisation of the probe in polymerase chain reaction or ligase chain reaction using an array as defined in claim 51.

86. A method for characterising cDNA, which method comprises:

(a) cutting a sample comprising a population of one or more cDNAs with a restriction endonuclease and isolating fragments bearing one end of the cDNA whose restriction site is at a reference site proximal to the end of the cDNA;

(b) cutting the isolated fragments with a first sampling endonuclease at a first sampling site of known displacement from the reference site to generate a first and second sub-fragment, each comprising a sticky end sequence of predetermined length and unknown sequence, the first sub-fragment having the end of the cDNA;

(c) sorting either the first or second sub-fragments into sub-populations according to their sticky end sequence and recording the sticky end sequence of each sub-population as the first sticky end;

(d) cutting the sub-fragments in each sub-population with a second sampling endonuclease, which is the same as or different from the first sampling endonuclease, at a second sampling site of known displacement from the first sampling site to generate from each sub-fragment a further sub-fragment comprising a second sticky end sequence of predetermined length and unknown sequence; and

(e) determining each second sticky end sequence;

wherein the aggregate length of the first and second sticky end sequences of each sub-fragment is from 6 to 10, the sequences and relative positions of the reference site and first and second sticky ends characterise the or each cDNA, the first sampling endonuclease binds to a first recognition site and cuts at the first sampling site at a predetermined displacement from the restriction site of the restriction endonuclease, and wherein the first and/or second recognition sites are provided in first and/or second adaptor oligonucleotides from an array according to claim 58, and hybridised to the restriction site of the isolated fragments.

87. A method for sequencing nucleic acid, which comprises:

(a) obtaining a target nucleic acid population comprising nucleic acid fragments in which each fragment is present in a unique amount and bears at one end a sticky end sequence of predetermined length and unknown sequence,

(b) protecting the other end of each fragment, and

(c) sequencing each of the fragments by

(i) contacting the fragments under hybridisation conditions in the presence of a ligase with an array according to claim 58, the base sequence of which having the same predetermined length as the sticky end sequence, the array containing all possible base sequences of that predetermined length;

removing any ligated adaptor oligonucleotide and recording the quantity of any ligated adaptor oligonucleotide by releasing the mass label and identifying the released mass label by mass spectrometry;

(ii) contacting the ligased adaptor oligonucleotides with a sequencing enzyme which binds to the recognition site and cuts the fragment to expose a new sticky end sequence which is contiguous with or overlaps the previous sticky end sequence; and

(iii) repeating steps (i) and (ii) for a sufficient number of times and determining the sequence of the fragment by comparing the quantities recorded for each sticky end sequence.

63. A method for determining hybridisation of an array of probes with a target nucleic acid, which method comprises

(a) contacting target nucleic acid with each hybridisation probe of the array under conditions to hybridise the probe to the target nucleic acid, and optionally removing unhybridised material, wherein each probe comprises a mass label linked to a known base sequence of predetermined length wherein each mass label comprises a photoexcitation group;

and

(b) identifying the hybridised probe by mass spectometry.

64. The method according to claim 63, wherein each mass label is cleavably linked to its respective known base sequence and each hybridised probe is cleaved to release the mass label, which released label is identified using a mass spectrometer.

68. The method according to claim 63, wherein the or each sample is analysed by matrix-assisted laser desorption ionization mass spectrometry.

69. The method according to claim 63, wherein the predetermined length of the base sequence is from 2 to 25.

70. The method according to claim 63, wherein the or each mass label is cleavably linked to the known base sequence by a collision-cleavable, photo-cleavable, chemically-cleavable or thermally-cleavable link.

71. The method according to claim 64, wherein the link is cleaved in the mass spectometer.

72. The method according to claim 71, wherein cleavage of the link is induced by laser photocleavage.

73. The method according to claim 71, wherein cleavage of the link is induced by collision.

74. The method according to claim 64, wherein each mass label is negatively-charged under ionisation conditions.

75. The method according to claim 64, wherein the mass labels and known base sequences are not separated before entry into the mass spectrometer.

76. The method according to claim 63, wherein the known base sequence comprises a sticky end of an adaptor oligonucleotide containing a recognition site for a restriction endonuclease which cuts at a predetermined displacement from the recognition site.

77. The method according to claim 63, wherein the known base sequence has linked thereto a plurality of identical mass labels.

78. The method according to claim 63, wherein the photo-excitation group is an excitable ionisation agent and is suitable for performing matrix-assisted laser desorption ionisation.

79. The method according to claim 63, wherein the photo-excitation group is selected from nicotinic acid, sinapinic acid or cinnamic acid.

80. The method according to claim 63, which is carried out in-line.

81. The method according to claim 63, wherein the mass label is resolvable in mass spectrometry from the known base sequence.

65. A method for determining hybridisation of a probe by mass spectrometry of a mass label optionally together with a known base sequence, using a hybridisation probe, comprising a mass label linked to a known base sequence of predetermined length, wherein the mass label comprises a photo-excitation group.

67. The method according to claim 65, wherein the mass label is cleavably linked to its respective known base sequence and the hybridised probe is cleaved to release the mass label, which released label is identified using a mass spectometer.

66. A method for determining hybridisation of a probe with a target nucleic acid, which method comprises

(a) contacting target nucleic acid with a hybridisation probe, which comprises a mass label linked to a known base sequence of predetermined length, under conditions to hybridise the probe to the target nucleic acid and optionally removing unhybridised material wherein the mass label comprises a photo-excitation group;

and

(b) identifying the hybridised probe by mass spectrometry.