Compositions and methods for labeling of nucleic acid molecules
First Claim
Patent Images
1. A composition for use in labeling one or more nucleic acid molecules, said composition comprising:
- a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes and are reduced or substantially reduced in RNase H activity; and
b) one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′
7′
-dimethoxy-4′
5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′
dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′
-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR.
2 Assignments
0 Petitions
Accused Products
Abstract
The present invention is generally related to compositions, kits and methods for labeling nucleic acid molecules using reverse transcriptases, preferably multi-subunit reverse transcriptases such as ASLV reverse transcriptases. Specifically, the invention relates to methods, kits and compositions for fluorescently labeling nucleic acid molecules during nucleic acid synthesis. The labeled nucleic acid molecules produced in accordance with the invention are particularly suited as labeled probes for nucleic acid detection and diagnostics.
-
Citations
41 Claims
-
1. A composition for use in labeling one or more nucleic acid molecules, said composition comprising:
-
a) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes and are reduced or substantially reduced in RNase H activity; and b) one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′
7′
-dimethoxy-4′
5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′
dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′
-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR. - View Dependent Claims (2, 3, 4, 5, 6, 18, 19, 20, 21, 22, 23, 24, 25)
-
-
7. A method for reverse transcription of one or more nucleic acid molecules, said method comprising:
-
(a) mixing one or more nucleic acid templates with; (i) one or more enzymes having reverse transcriptase activity, wherein said enzymes are multi-subunit enzymes and are reduced or substantially reduced in RNase H activity; and (ii) one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′
7′
-dimethoxy-4′
5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′
dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′
-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR; and(b) incubating said mixture under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein at least one of said first nucleic acid molecules is fluorescently labeled. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 26, 27, 28, 29, 30, 31, 32, 33)
-
-
16. A kit for use in labeling one or more nucleic acid molecules, said kit comprising one or more enzymes having reverse transcriptase activity, and one or more deoxynucleotides labeled with a fluorescent label selected from the group consisting of 5-carboxyfluorescein (FAM), 2′
- 7′
-dimethoxy-4′
5-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N′
,N′
-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′
dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Cyanine, 5-(2′
-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), Cy3, Cy5, ChromaTide, BODIPY-FL and BODIPY-TMR, wherein said enzymes are multi-subunit enzymes and are reduced or substantially reduced in RNase H activity. - View Dependent Claims (17, 34, 35, 36, 37, 38, 39, 40, 41)
- 7′
Specification