Methods for detecting nucleic acid analyte

• US 20060134666A1
• Filed: 11/10/2005
• Published: 06/22/2006
• Est. Priority Date: 05/19/1999
• Status: Abandoned Application

• Current Assignee: Harold G. Craighead, Jonas Korlach, Mathieu Foquet, Michael Levene, Stephen Turner, Watt W. Webb
• Original Assignee: Harold G. Craighead, Jonas Korlach, Mathieu Foquet, Michael Levene, Stephen Turner, Watt W. Webb
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Application Number:
  US11/271,033
• Publication Number:
  US 20060134666A1
• 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 detecting an analyte comprising the steps of:

(a) anchoring said analyte to a nucleic acid template;

(b) conducting a nucleic acid polymerase reaction to produce labeled polyphosphate, said reaction comprising the reaction of said template, a primer, at least one terminal phosphate-labeled nucleotide, and a nucleic acid polymerase; and

(c) analyzing said labeled polyphosphate.

63. The method of claim 62, wherein said primer is a nuclease resistant primer.

64. The method of claim 63, wherein the nucleic acid polymerase reaction further includes an enzyme having 3′

-5′

exonuclease activity.

65. The method of claim 62, wherein the analyte is DNA or RNA.

66. The method of claim 63, wherein said primer comprises modified ribonucleotides or modified deoxyribonucleotides.

67. The method of claim 62, further comprising the step of characterizing said analyte.

68. The method of claim 67, further comprising the step of quantifying said analyte.

69. The method of claim 62, wherein said analyte is a synthetic polymer.

70. The method of claim 62, wherein said analyte is anchored to said nucleic acid template by non-covalent binding, or by one or more covalent bonds.

71. The method of claim 62, wherein said nucleic acid polymerase is a DNA polymerase or an RNA polymerase.

72. The method of claim 63, wherein said primer comprises thiol-group-containing ribonucleotides or or deoxyribonucleotides.

73. The method of claim 62, wherein said nucleic acid template and said primer are switched and it is said primer that is anchored to the analyte.

74. The method of claim 62, wherein said nucleic acid template and said primer are part of a DNA hairpin, and said DNA hairpin is anchored to said analyte in said anchoring step.

75. The method of claim 62, wherein at least one terminal phosphate-labeled nucleotide includes two or more phosphate groups in the polyphosphate chain.

76. The method of claim 62, wherein at least one terminal phosphate-labeled nucleotide includes three or more phosphate groups in the polyphosphate chain.

77. The method of claim 62, wherein the labels in at least one terminal phosphate-labeled nucleotide are enzyme-activatable labels selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

78. The method of claim 62, wherein said terminal phosphate-labeled nucleotides carry distinct labels.

79. The method of claim 78, wherein the presence of an analyte is determined by the ratio of distinct labels produced.

80. The method of claim 62, wherein at least one terminal phosphate-labeled nucleotides are deoxy nucleotides and carry different labels.

81. The method of claim 62, wherein at least one terminal-phosphate-labeled nucleotide is represented by the formula:

Base unit-S—

Y—

(PO₃)_n-Label moiety

wherein the base unit is a nitrogen-containing heterocyclic base, S is a carbocyclic moiety or sugar moiety, Y is an oxygen or sulfur atom, PO₃ is a phosphate group and n is an integer of 1 or greater, L is an enzyme-activatable label containing a sulfhydryl group or an amino group suitable for forming a phosphate ester or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide; and

L is a phosphorylated label which preferably becomes independently detectable when the phosphate is removed.

82. The method of claim 81, wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, adenine, and analog thereof.

83. The method of claim 81, wherein Y is an oxygen and n is an integer of 2 or greater.

84. The method of claim 81, wherein S is a sugar moiety and L is a phosphorus-containing label.

85. The method of claim 81, wherein said enzyme-activatable label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

86. The method of claim 81, wherein the sugar moiety is selected from the group consisting of a ribosyl, a deoxyribosyl, an acyclic, and a modified sugar.

91. The method of claim 62, wherein said analyzing step includes (a) reacting said labeled polyphosphate with one or more additional enzymes to produce a detectable species characteristic of said analyte and (b) detecting said detectable species.

92. The method of claim 62, further including the step of separating any nucleic acid template not anchored by said analyte before said conducting step.

93. The method of claim 91, wherein said reacting step and said conducting step are carried out simultaneously.

94. The method of claim 91, wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, oxidation/reduction potential, mass change, and combinations thereof.

95. The method of claim 91, wherein said detectable species is produced in amounts substantially proportional to the amount of analyte.

97. The method of claim 81, wherein n is an integer of 3 or greater.

98. The method of claim 85, wherein the enzyme-activatable label is a fluorogenic moiety comprising fluorescein.

87. A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:

(a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;

(b) conducting a DNA polymerase reaction to produce uniquely labeled polyphosphates;

said reaction comprising the reaction of said templates, nuclease resistant primers complementary to said specific target sequence of each of said multiple analytes, two or more terminal phosphate-labeled nucleotides having 2 or more phosphate groups in the polyphosphate chain and each bearing a different label, a DNA polymerase and an enzyme having 3′

-5′

exonuclease activity; and

(c) detecting the labeled polyphosphates.

88. The method of claim 87, wherein the terminal phosphate-labeled nucleotides have 3 or more phosphate groups.

89. The method of claim 87, wherein the analyte is DNA or RNA.

90. The method of claim 87, wherein said primer comprises modified ribonucleotides or modified deoxyribonucleotides.

96. The method of 62, wherein one or more additional detection reagents are added in said polymerase reaction of said conducting step, and said additional detection reagents are capable of a response that is detectably different from said labeled polyphosphate.

99. A method of detecting and characterizing multiple analytes in a reaction compartment, comprising the steps of:

(a) anchoring a unique template nucleic acid sequence to each of said analytes;

(b) anchoring said analytes to the surface of said reaction compartment;

(c) conducting a DNA polymerase reaction to produce labeled polyphosphate;

said reaction comprising the reaction of the unique template sequence of one of said analytes, a nuclease resistant primer complementary to said unique template sequence, at least one terminal phosphate-labeled nucleotides having 2 or more phosphate groups in the polyphosphate chain, a DNA polymerase and an enzyme having 3′

-5′

exonuclease activity;

(d) detecting said labeled polyphosphate;

(e) washing off the unanchored components; and

(f) repeating steps (a) to (d) with a nuclease resistant primer complementary to another unique template sequence of a different analyte until all the analytes are analyzed.

100. The method of claim 99, wherein said at least one terminal phosphate labeled nucleotides have 3 or more phosphate groups in the polyphosphate chain.

101. The method of claim 99, wherein said detecting step includes:

(a) permitting said labeled polyphosphate to react with one or more additional enzyme to produce a detectable species; and

(b) detecting said detectable species.

102. A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:

(a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;

(b) conducting a DNA polymerase reaction to produce labeled polyphosphates, said reaction comprising the reaction of said templates, primers complementary to said specific template sequence, two or more terminal phosphate-labeled nucleotides with different labels, a DNA polymerase and an enzyme having 3′

-5′

exonuclease activity;

(c) permitting said labeled polyphosphates to react with one or more additional enzymes to produce detectable species unique to each of said analytes; and

(d) detecting said detectable species.

103. A method of sequencing a target nucleic acid, comprising:

providing a reaction mixture comprising the target nucleic acid, a primer, a polymerase enzyme, and one or more labeled nucleotides or nucleotide analogs, wherein the target nucleic acid and/or the primer is immobilized upon a substrate;

sequentially optically detecting incorporation of at least first and second labeled nucleotides or nucleotide analogs in a nascent nucleic acid strand by an individual polymerase enzyme; and

identifying the first and second nucleotides or nucleotide analogs incorporated into the nascent nucleic acid strand.

105. The method of claim 103 or 104, wherein the primer is coupled to the substrate.

106. The method of claim 103 or 104, wherein the reaction mixture comprises a plurality of different labeled nucleotides or nucleotide analogs.

107. The method of claim 103 or 104, wherein the reaction mixture comprises at least four different nucleotides or nucleotide analogs.

108. The method of claim 103 or 104, wherein the four different nucleotides or nucleotide analogs each comprises a different fluorescent label.

109. The method of claim 103 or 104, wherein the at least first and second labeled nucleotide or nucleotide analogs each comprises a fluorescent label that is removed prior to incorporation of a subsequent labeled nucleotide or nucleotide analog.

110. The method of claim 109, wherein removal of the fluorescent label is carried out by photochemical cleavage of the fluorescent label from the incorporated nucleotide or nucleotide analog.

111. The method of claim 103 or 104, wherein the at least first and second labeled nucleotides or nucleotide analogs each comprises a fluorescent label that is removed during incorporation of the first nucleotide or nucleotide analog into the nascent nucleic acid strand.

112. The method of claim 103 or 104, wherein the fluorescent label comprises at least one member of a FRET pair.

113. The method of claim 103 or 104, wherein an individual polymerase enzyme-template polynucleotide complex immobilized upon the substrate is optically resolvable from other polymerase enzyme-template polynucleotide complexes immobilized on the substrate.

114. The method of claim 103 or 104, wherein the template polynucleotide is coupled to the substrate.

115. The method of claim 103 or 104, wherein the polymerase enzyme is coupled to the substrate.

116. The method of claim 103 or 104, further comprising separately sequentially optically detecting incorporation of labeled nucleotides or nucleotide analogs in nascent nucleic acid strands by a plurality of individual polymerase enzymes.

104. A method of sequencing a target nucleic acid, comprising:

providing a reaction mixture comprising the target nucleic acid, a primer, a polymerase enzyme, and one or more labeled nucleotides or nucleotide analogs, wherein the target nucleic acid and/or the primer is immobilized upon a substrate;

sequentially optically detecting incorporation of at least first and second labeled nucleotides or nucleotide analogs into an individual nascent nucleic acid strand by the polymerase enzyme; and

identifying the first and second nucleotides or nucleotide analogs incorporated into the nascent nucleic acid strand.

117. A method of sequencing a target nucleic acid, comprising optically detecting sequential incorporation of nucleotides or nucleotide analogs into a nascent nucleic acid strand by an individual polymerase enzyme complexed with the template polynucleotide, wherein the template polynucleotide is coupled to a substrate.

118. The method of claim 117, wherein the template polynucleotide is coupled to the substrate through a primer that is bound to the substrate.

119. A method for sequencing a target nucleic acid, comprising:

(a) providing a complex comprising a polymerase and a target nucleic acid such that said polymerase is capable of incorporating a nucleotide or nucleotide analog complementary to a nucleotide on the target nucleic acid, wherein the complex is immobilized on a substrate;

(b) providing a plurality of labeled nucleotides or nucleotide analogs to the complex to effect incorporation of a labeled nucleotide or analog into a nascent strand that is complementary to the target nucleic acid;

(c) optically detecting the incorporated nucleotide or analog by sensing its detectable label, wherein said detecting is performed by applying radiation through the complex at a spatial location of the complex where said label is expected to occur;

(d) repeating, with the complex immobilized on the substrate, steps (b) through (c) so that a plurality of nucleotides incorporated is identified and, as a result, a sequence of the target nucleic acid is determined.

120. The method of claim 119, wherein the complex further comprises a primer oriented in such a way to permit incorporation of labeled nucleotides or analogs complementary to the target nucleic acid.

121. The method of claim 120, wherein the primer is coupled to the substrate.

122. The method of claim 119, wherein the plurality of labeled nucleotides or nucleotide analogs are of the same type.

123. The method of claim 119, wherein the plurality of labeled nucleotides or nucleotide analogs are of different types.

124. The method of claim 119, wherein the incorporated nucleotide or nucleotide analog comprises a fluorescent label that is removed prior to incorporation of a subsequent labeled nucleotide or nucleotide analog.

125. The method of claim 124, wherein removal of the fluorescent label is carried out by photochemical cleavage of the fluorescent label from the incorporated nucleotide or nucleotide analog.