Fluorescent Nucleobase Conjugates Having Anionic Linkers

• US 20090092986A1
• Filed: 09/29/2008
• Published: 04/09/2009
• Est. Priority Date: 10/11/2000
• Status: Active Grant

• Current Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: TAING, Meng, KHAN, Shaheer H., MENCHEN, Steven M., ROSENBLUM, Barnett B.
• Granted Patent: US 7,687,236 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

Claims

1-71. -71. (canceled)

72. A nucleic acid sequencing kit comprising at least one labeled nucleoside triphosphate comprising a conjugate comprising a dye labeled nucleobase of the form:

(1) B-L-D, wherein B is a nucleobase, L is an anionic linker, and D comprises at least one fluorescent dye that comprises a xanthene, a rhodamine or a fluorescein, or

(2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers such that at least one of L1 and L2 is an anionic linker, and D1 and D2 are members of a fluorescent donor/acceptor pair, such that one of D1 and D2 is a donor dye capable of absorbing light at a first wavelength and emitting energy in response thereto, and the other of D1 and D2 is an acceptor dye capable of absorbing energy emitted by the donor dye and fluorescing at a second wavelength in response thereto, and at least one of D1 and D2 comprises a xanthene, a rhodamine or a fluorescein,

wherein L or at least one of L1 and L2 comprises at least one anionic phosphate or anionic phosphate and one or more of the following components;

a 3′

-extendable primer,

a polymerase enzyme,

one or more 3′

extendable nucleotides which are not labeled with conjugate, and/or a buffering agent.

73. The kit of claim 72 wherein at least one labeled nucleoside triphosphate is nonextendable.

74. The kit of claim 72 which comprises four different labeled nucleoside triphosphates which are complementary to A, C, T and C, and each of which contains a distinct conjugate.

75. The kit of claim 74 wherein the four different labeled nucleoside triphosphates are nonextendable.

76. The kit of claim 74 wherein the four different labeled nucleoside triphosphates are extendable ribonucleoside triphosphates.

77. The kit of claim 72 wherein the conjugate has the form B-L1-D1-L2-D2.

78. The kit of claim 77 wherein the donor dyes in the four different labeled nucleoside triphosphates are the same.

79. The kit of claim 78 wherein the donor dye is an orthocarboxyfluorescein.

80. The kit of claim 78 wherein the donor dye is a 4,7-dichloro-orthocarboxyfluorescein.

86. The kit of claim 77 wherein L2 is an anionic linker and L1 is not an anionic linker.

87. The kit of claim 77 wherein L2 comprises a carboxylic acid moiety.

88. The kit of claim 87 wherein the carboxylic acid moiety is a carboxy benzene moiety.

89. The kit of claim 72 wherein the dye-labeled nucleobase is of the form B-L-D.

90. The kit of claim 89 wherein L comprises a sulfonic acid moiety.

91. The kit of claim 89 wherein L comprises a sulfonated benzene moiety.

92. The kit of claim 89 wherein L comprises an anionic phosphate moiety.

93. The kit of claim 92 wherein the anionic phosphate moiety is a phosphate diester moiety, and the phosphorus atom of the phosphate diester moiety is located in L within a chain of linker atoms that connect B to D.

94. The kit of claim 89 wherein L comprises an anionic phosphonate moiety.

95. The kit of claim 94 wherein the anionic phosphonate moiety is a phosphonate monoester moiety, and the phosphorus atom of the phosphonate monoester moiety is located in L within a chain of linker atoms that connect B to D.

96. The kit of claim 89 wherein L comprises a carboxylic acid moiety.

97. The kit of claim 96 wherein the carboxylic acid moiety is a carboxyl benzene moiety.

98. The kit of claim 89 wherein L comprises 4 to 20 chain atoms.

99. The kit of claim 89 wherein D comprises at least one fluorescein or rhodamine.

100. The kit of claim 72 wherein B comprises adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine.

81-85. -85. (canceled)