Method for sequencing nucleic acid molecules

• US 20050164255A1
• Filed: 12/15/2004
• Published: 07/28/2005
• Est. Priority Date: 05/19/1999
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Granted Patent: US 7,052,847 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Claims

1-61. -61. (canceled)

62. A method of sequencing a target nucleic acid molecule comprising:

(a) providing a mixture comprising the target nucleic acid molecule, a primer complementary to the target nucleic acid molecule, a nucleic acid polymerizing enzyme, and a plurality of types of nucleotides or nucleotide analogs to be incorporated into a growing nucleic acid strands at an active site complementary to the target nucleic acid;

(b) subjecting the mixture of step (a) to a polymerization reaction under conditions suitable for formation of the growing nucleic acid strands by template-directed extension of primer; and

(c) identifying a time sequence of incorporation of nucleotides or nucleotide analogs into the growing nucleic acid strands at the active site while the template-directed extension of primer is taking place, and thereby determining the sequence of said target nucleic acid.

64. A method according to claim 62 or 63, wherein said identifying is performed without interrupting the template-directed extension of primer.

65. A method according to claim 62 or 63, wherein said identifying is performed without adjusting components of the mixture prior to reading the next base sequence.

66. A method according to claim 62 or 63, wherein said time sequence of incorporation is a time sequence of at least 10,000 nucleotides or nucleotide analogs.

67. A method according to claim 62 or 63, wherein said time sequence of incorporation is a time sequence of at least 3 nucleotides or nucleotide analogs.

68. A method according to claim 62 or 63, wherein the polymerization reaction is performed by a nucleic acid polymerizing enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase, and a mixture thereof.

69. A method according to claim 62 or 63, wherein the nucleic acid polymerizing enzyme is a thermostable polymerase or a thermodegradable polymerase.

70. A method according to claim 62 or 63, wherein the target nucleic acid molecule is selected from the group consisting of double-stranded DNA, single-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, and RNA hairpins.

71. A method according to claim 62 or 63, wherein the nucleotide analogs are selected from the group consisting of a ribonucleotide, a deoxyribonucleotide, a modified ribonulcleotide, a modified deoxyribonucleotide, a peptide nucleotide, a modified peptide nucleotide, and a modified phosphate-sugar backbone nucleotide.

72. A method according to claim 62 or 63, wherein the nucleotides or nucleotide analogs further comprise a label.

73. A method according to claim 72, wherein the label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, scattering or fluorescent nanoparticles, Raman signal generating moieties, and electrochemical detection moieties.

74. A method according to claim 72, wherein the label is attached to the nucleotide or nucleotide analog at a base, sugar moiety, alpha phosphate, beta phosphate, or gamma phosphate of said nucleotide or nucleotide analog.

75. A method according to claim 72, wherein each of the plurality of types of nucleotides or nucleotide analogs have different labels which are distinguished from one another during said identifying.

76. A method according to claim 72, wherein three or less of the plurality of types of nucleotides or nucleotide analogs have a different label.

77. A method according to claim 72, wherein the different types of nucleotides or nucleotide analogs have the same label but are distinguished by different properties due to a presence of base fluorophores, quenched fluorophores, or fluorogenic nucleotides or nucleotide analogs.

78. A method according to claim 62, wherein the nucleic acid polymerizing enzyme carries a label and said identifying is carried out by detecting interaction between the label and the nucleotide or nucleotide analog.

79. A method according to claim 78, wherein the label is a fluorescence resonance energy transfer donor or acceptor.

80. A method according to claim 62 or 63, wherein said identifying is carried out by optical procedures selected from the group consisting of far-field microspectroscopy, near-field microspectroscopy, evanescent wave or wave guided illumination, nanostructure enhancement, and combinations thereof.

81. A method according to claim 62 or 63, wherein said identifying is carried out by single photon excitation, multiphoton excitation, fluorescence resonance energy transfer, photoconversion or a combination thereof.

82. A method according to claim 62 or 63, wherein said identifying is achieved by spectral wavelength discrimination, measurement and separation of fluorescence lifetimes, fluorophore identification, background suppression, or a combination thereof.

83. A method according to claim 82, wherein fluorophore identification or background suppression utilizes fast switching between excitation modes and illumination sources, and combinations thereof.

84. A method according to claim 62 or 63, wherein said target nucleic acids are attached to a support.

85. The method of claim 62 or 63, wherein said target nucleic acid is hybridized to an oligonulceotide which is attached to a support.

86. The method of claim 68, wherein said DNA polymerizing enzyme is attached to a support.

87. A method according to claim 62 or 63, wherein said identifying is carried out by reducing background noise resulting from free nucleotides or nucleotide analogs.

88. A method according to claim 87, wherein said identifying comprises directing activating radiation to a region substantially corresponding to the active site and detecting the nucleotide or nucleotide analog polymerized at the active site.

89. A method according to claim 87, wherein said identifying distinguishes nucleotides or nucleotide analogs polymerized at the active site from free nucleotides or nucleotide analogs.

90. A method according to claim 87, wherein said identifying is carried out in a confined region proximate to the active site.

91. A method according to claim 90, wherein said identifying is carried out in a nanostructure.

92. A method according to claim 91, wherein the nanostructure is a punctuate, acicular, or resonant nanostructure which enhances said detecting.

93. A method according to claim 90, wherein nucleotides or nucleotide analogs that are not polymerized at the active site move rapidly through a microstructure to and from the confined region.

94. A method according to claim 93, wherein the microstructure comprises:

a plurality of channels to direct different nucleotides or nucleotide analogs to the confined region and a discharge channel to permit materials to be removed from the confined region, and the nanostructure comprises;

a housing defining the confined region and constructed to facilitate said identifying.

95. A method according to claim 87, wherein said identifying is carried out by electromagnetic field enhancement with electromagnetic radiation being enhanced proximate to an object with a small radius of curvature adjacent to the active site.

96. A method according to claim 87, wherein said identifying is carried out by near-field illumination of cavities in which the primed target nucleic acid molecule is positioned.

97. A method according to claim 87, wherein said identifying is carried out with optical fibers proximate to the active site.

98. A method according to claim 87, wherein said identifying and said reducing background is carried out by time gated delay of photon detection.

63. A method of sequencing a target nucleic acid molecule comprising:

subjecting the target nucleic acid molecule to a polymerization reaction to yield a growing nucleic acid strand that is complementary to the target nucleic acid molecule in the presence of a reaction mixture of plurality of types of nucleotides or nucleotide analogs; and

identifying a time sequence of incorporation of said plurality of types of nucleotides or nucleotide analogs in said reaction mixture into the growing nucleotide strand.