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US4965349A
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Method of synthesizing oligonucleotides labeled with ammonia-labile groups on solid phase supports
Assignee: Applied Biosystems LLC
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Filing: 1987-12-24
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Granted: 1990-10-23
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Expires: 2007-12-24
Abstract
Abstract The invention provides a novel cleavage reagent for hydrolysing base-labile linking groups between a solid phase support and oligonucleotides. The cleavage reagent comprises a lower alkyl alcohol, water, and a non-nuccleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1, respectively. An important property of the cleavage reagent is that it preserves the fluorescent characteristics of rhodamine dyes during cleavage, thereby making it possible to completely synthesize rhodamine-labeled oligonucleotides by solid phase methods. Rhodamine phosphoramidites are provided to further enhance the efficiency of synthesizing rhodamine-labeled oligonucleotides by such methods.
Inventors
- 👤 Sam L. Woo
- 👤 Steven M. Menchen
- 👤 Steven Fung
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 22:23
CPC Classifications
C; C07; C07H; C07H21/00; C07F; C07F9/00; C07F9/02; C07F9/06; C07F9/22; C07F9/24
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
67 words
A process of cleaving a base-labile linking group between an oligonucleotide and a solid phase support comprising the step of exposing the linking group to a cleavage reagent until the linking group is hydrolyzed, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1 by volume, respectively.
Claim 2
Dependent Claim
67 words
A process of cleaving a base-labile linking group between an oligonucleotide and a solid phase support comprising the step of exposing the linking group to a cleavage reagent until the linking group is hydrolyzed, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1 by volume, respectively.
Claim 3
Dependent Claim
42 words
The process of claim 1 wherein said lower alkyl alcohol is selected from the group consisting of methanol, ethanol, and propanol, and wherein said nonnucleophilic hindered alkylamine is selected from the group consisting of isopropylamine, t-butylamine, diethylamine, piperidine, t-amylamine, diisopropylamine, and dipropylamine.
Claim 4
Dependent Claim
13 words
The process of claim 2 wherein said linking group is a succinate ester.
Claim 5
Dependent Claim
21 words
The process of claim 3 wherein said cleavage reagent comprises methanol, water and t-butylamine in a ratio of about 1:2:1, respectively.
Claim 6
Dependent Claim
111 words
A process of synthesizing oligonucleotides labeled with an ammonia-labile group on a solid phase support, the method comprising the steps of: providing an oligonucleotide attached to a solid phase support by a base-labile linking group; attaching one or more ammonia-labile groups to the oligonucleotide to form an oligonucleotide labeled with an ammonia-labile group; and cleaving the linking group with a cleavage reagent so that the oligonucleotide labeled with an ammonia-labile group is freed from the solid phase support, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1 by volume, respectively.
Claim 7
Dependent Claim
111 words
A process of synthesizing oligonucleotides labeled with an ammonia-labile group on a solid phase support, the method comprising the steps of: providing an oligonucleotide attached to a solid phase support by a base-labile linking group; attaching one or more ammonia-labile groups to the oligonucleotide to form an oligonucleotide labeled with an ammonia-labile group; and cleaving the linking group with a cleavage reagent so that the oligonucleotide labeled with an ammonia-labile group is freed from the solid phase support, the cleavage reagent comprising a lower alkyl alcohol, water, and a non-nucleophilic hindered alkylamine containing from 3 to 6 carbon atoms in a ratio of about 1:1:1 to about 1:3:1 by volume, respectively.
Claim 8
Dependent Claim
26 words
The process of claim 5 wherein said linking group forms a covalent bond between the 3' hydroxyl group of said oligonucleotide and said solid phase support.
Claim 9
Dependent Claim
52 words
The process of claim 6 wherein (1) said lower alkyl alcohol is selected from the group consisting of methanol, ethanol, and propanol, (2) said non-nucleophilic hindered alkylamine is selected from the group consisting of isopropylamine, t-butylamine, diethylamine, piperidine, t-amylamine, diisopropylamine, and dipropylamine, and (3) said base-labile linking group is a succinate ester.
Claim 10
Dependent Claim
14 words
The process of claim 7 wherein said ammonia-labile group is a rhodamine dye molecule.
Claim 11
Dependent Claim
21 words
The process of claim 8 wherein said cleavage reagent comprises methanol, water, and t-butylamine in a ratio of about 1:2:1, respectively.
Claim 12
Dependent Claim
28 words
The process of claim 8 wherein said step of attaching one or more rhodamine dye molecules includes reacting a rhodamine phosphoramidite with the 5' hydroxyl of said oligonucleotide.
Claim 13
Dependent Claim
14 words
The process of claim 5 wherein said ammonia-labile group is a rhodamine dye molecule.
References Cited (Backward Citations)
US4659774A
Cited
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