Terminal-phosphate-labeled nucleotides

• US 20060160113A1
• Filed: 12/01/2005
• Published: 07/20/2006
• 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:
  US11/292,746
• Publication Number:
  US 20060160113A1
• 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 assaying for the rate of an enzyme catalyzed nucleoside monophosphate transfer from a terminal-phosphate-labeled nucleoside polyphosphate to detect the activity of said enzyme or said terminal-phosphate-labeled nucleoside polyphosphate, said method comprising:

a) conducting said enzyme catalyzed nucleoside monophosphate transfer from a terminal-phosphate-labeled nucleoside polyphosphate reaction in reaction buffer comprising a divalent metal ion, thereby increasing the rate of said reaction over the rate of said reaction in the absence of the divalent metal ion.

63. The method of claim 62, wherein the enzyme is selected from a nucleic acid polymerase, a ligase, primase, or nucleotide hydrolase.

64. The method of claim 62, wherein the enzyme is a nucleic acid polymerase or a primase.

65. The method of claim 63, wherein the nucleic acid polymerase is selected from DNA polymerase, RNA polymerase, reverse transcriptase, or terminal transferases.

66. The method of claim 63, wherein the nucleic acid polymerase is selected from DNA polymerase, RNA polymerase, or reverse transcriptase.

67. The method of claim 63, wherein the polymerase is selected from Klenow fragment, Sequenase, Taq DNA polymerase, P. furiosis (Strategene), AMV-reverse transcriptase, MMLV-reverse transcriptase, and HIV-reverse transcriptase.

68. The method of claim 62, wherein the divalent metal ion is magnesium.

69. The method of claim 62, wherein the divalent metal ion is present at a concentration of micromolar range.

70. The method of claim 62, wherein the divalent metal ion is present at a concentration of about 25 mM.

71. The method of claim 62, wherein an additional metal salt other than magnesium is also present with the terminal-phosphate labeled nucleoside polyphosphate.

72. The method of claim 71, wherein said additional metal salt is a sodium salt.

73. The method of claim 71, wherein said additional metal salt is present at a concentration of micromolar range.

74. The method of claim 62, further comprising conducting said reaction in the presence of a metal ion buffer to modulate the concentration of free metal ion.

75. A nucleic acid detection reagent comprising:

a) one or more terminal-phosphate-labeled nucleotide according to the following formula;

wherein

P is a phosphate (PO₃) or a derivative thereof, and n is 1 or greater;

Y is an oxygen or sulfur atom;

the base unit (B) is a nitrogen-containing heterocyclic base;

S is a carbocyclic moiety or sugar moiety;

P—

Label moiety (L) is a phosphorylated label with a linker between L and P,

wherein L is a label containing a hydroxyl group, a sulfhydryl group, a haloalkyl group or an amino group suitable for forming a phosphate ester, a thioester, an alkylphosphonate or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide;

b) one or more DNA polymerase, RNA polymerase, or reverse transcriptase; and

c) reaction buffer containing a divalent metal ion salt.

76. The nucleic acid detection reagent of claim 75, wherein L is a label containing a hydroxyl group, a sulfhydryl group or an amino group suitable for forming a phosphate ester, thioester, an alkylphosphonate or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide.

80. The reagent of claim 75 or claim 77, wherein said sugar moiety is selected from the group consisting of ribosyl, deoxyribosyl, carbocyclic, and other modified sugars.

81. The reagent of claim 75 or claim 77, wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, adenine, and analogs thereof.

82. The reagent of claim 75 or claim 77, wherein said label is selected from the group consisting of chemiluminescent compounds, fluorescent compounds, colored dyes, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

83. The reagent of claim 75 or claim 77, wherein n is an integer of 2 or greater.

77. A nucleic acid detection reagent comprising:

a) one or more terminal-phosphate-labeled nucleotide according to the following formula;

wherein

P is a phosphate (PO₃) or a derivative thereof, and n is 1 or greater;

Y is an oxygen or sulfur atom;

the base unit (B) is a nitrogen-containing heterocyclic base;

S is a carbocyclic moiety or sugar moiety;

P—

Label moiety (L) is a phosphorylated label with a linker between L and P,

wherein L is a label containing a hydroxyl group, a sulfhydryl group, a haloalkyl group or an amino group suitable for forming a phosphate ester, a thioester, an alkylphosphonate or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide;

b) one or more DNA polymerase, RNA polymerase, or reverse transcriptase;

c) reaction buffer containing a divalent metal ion; and

d) a metal ion binding buffer.

78. The nucleic acid detection reagent of claim 77, wherein L is a label containing hydroxyl group, a sulfhydryl group or an amino group suitable for forming a phosphate ester, a thioester, an alkylphosphonate or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide.

79. The nucleic acid detection reagent of claim 77, wherein the linker between L and P comprises an NH group.

84. A terminal-phosphate labeled nucleoside polyphosphate of the following formula:

85. The terminal-phosphate labeled nucleoside polyphosphate of claim 84, wherein said sugar moiety is selected from the group consisting ribosyl;

deoxyribosyl, carbocyclic, and other modified sugars.

86. The terminal-phosphate labeled nucleoside polyphosphate of claim 84, wherein said base is selected from the group consisting of uracil, thymine, cytosihe, guanine, adenine, and analogs thereof.

87. A divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of following structure:

Label-NPP-(Divalent Metal Ion)_x,

wherein

Label is a detectable moiety connected to NPP with or without a linker;

NPP is a nucleoside polyphosphate with two or more phosphates; and

x is 1 or more.

88. The divalent metal ion complex of claim 87, wherein NPP is a nucleoside polyphosphate with three or more phosphates.

89. The divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of claim 87, wherein x is 1.

90. The divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of claim 87, wherein the nucleoside-polyphosphate is a natural or a modified nucleoside.

91. The divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of claim 87, wherein L is connected to the NPP through a linker of structure x-Z-y.

92. The divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of claim 91, wherein x-Z-y as a unit comprises an NH group.

93. A nucleic acid detection reagent comprising:

a) at least one divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of the following formula;

Label-NPP-(Divalent Metal Ion)_x,

wherein

Label is a detectable moiety linked to NPP with or without a linker;

NPP is a nucleoside polyphosphate with two or more phosphates;

and x is 1 or more; and

b) a nucleic acid polymerase.

94. The reagent of claim 93 wherein NPP is a nucleoside polyphosphate with three or more phosphates.

97. The reagent of claim 93 or claim 95, wherein said label is selected from the group consisting of chemiluminescent compounds, fluorescent compounds, colored dyes, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

95. A nucleic acid detection reagent comprising:

a) at least one divalent metal ion complex of a terminal-phosphate labeled nucleoside polyphosphate of the following formula;

Label-NPP-(divalent metal ion)_x

wherein

Label is a detectable moiety linked to NPP with or without a linker;

NPP is a nucleoside polyphosphate with two or more phosphates; and

x is 1 or more;

b) a nucleic acid polymerase; and

c) a metal-ion binding buffer.

96. The reagent of claim 95, wherein NPP is a nucleoside polyphosphate with three or more phosphates.