EP0272007A2
Expired / Lapsed
EP
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5- and 6- Succinimidyl - carboxylate isomers of rhodamine dyes
Assignee: Applied Biosystems Inc
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Filing: 1987-11-20
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Granted: —
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Expires: 2007-11-20
Abstract
Abstract Isomerically pure 5- and 6-succinimidylcarboxylates of rhodamine dyes are provided. A key feature in the preparation of the succinimidylcarboxylates is the use of stoichiometric amounts of di-N-succinimidylcarbonate and 4-dimethylaminopyridine for converting the 5- and/or 6-carboxyrhodamines to N-hydroxysuccinimide esters. In chain termination DNA sequencing procedures, using the pure isomeric forms of the activated rhodamines prevents generation of spurious sequence data because of the different electrophoretic mobilities of the isomers.
Inventors
- 👤 Steven M. Menchen
- 👤 Steven Fung
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 00:43
CPC Classifications
C; C09; C09B; C09B11/00; C09B11/04; C09B11/10; C09B11/24; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
13 words
An isomerically pure 5- or 6- succinimido N-carboxyl rhodamine dye and salts thereof.
Claim 2
Dependent Claim
13 words
An isomerically pure 5- or 6- succinimido N-carboxyl rhodamine dye and salts thereof.
Claim 3
Dependent Claim
118 words
A compound according to Claim 1, having the formula I shown hereinbefore, wherein R₁, R₄, R₅ and R₈ (which may be the same or different) are each hydrogen, halogen, or an alkyl, alkylether, or alkylthioether group having from 1 to 8 carbon atoms, R₂, R₃, R₆ and R₇ (which may be the same or different) are alkyl groups having from 1 or 8 carbon atoms, or wherein R₁ and R₂, R₃ and R₄, R ₅ and R₆, and R₇ and R₈, when taken in one or more pairs together constitute one or more alkylene bridging groups containing from 2 to 5 carbon atoms, and W₁, W₂, and W₃ are hydrogen or chloro, and B is an anionic group.
Claim 4
Dependent Claim
37 words
A compound according to Claim 2, wherein the groups R₁ to R₈ inclusive are taken in pairs as alkylene bridging groups each containing 3 carbon atoms, W₁, W₂ and W₃ are each hydrogen, and B is carboxyl.
Claim 5
Dependent Claim
30 words
A compound according to Claim 2, wherein R₁, R₄, R₅, R₈, W₁, W₂, and W₃ are each hydrogen, R₂, R₃, R₆, and R₇ are each methyl, and B is carboxyl.
Claim 6
Dependent Claim
18 words
A compound according to any of Claims 1 to 4, for use in the fluorescent labelling of nucleotides.
Claim 7
Independent Claim
30 words
A method of forming an N-hydroxysuccinimide ester of a rhodamine dye carboxylic acid comprising the step of: reacting the rhodamine dye carboxylic acid with equivalent amounts of di-N-succinimidylcarbonate and 4-dimethylaminopyridine.
Claim 8
Independent Claim
30 words
A method of forming an N-hydroxysuccinimide ester of a rhodamine dye carboxylic acid comprising the step of: reacting the rhodamine dye carboxylic acid with equivalent amounts of di-N-succinimidylcarbonate and 4-dimethylaminopyridine.
Claim 9
Dependent Claim
15 words
The method of claim 6 wherein the reaction is conducted in an aprotic polar solvent.
Claim 10
Dependent Claim
13 words
The method of claim 7 wherein the reaction is conducted at room temperature.
Claim 11
Dependent Claim
52 words
The method of claim 6, 7, or 8 wherein said rhodamine carboxylic acid is selected from a group consisting of isomerically pure 5-carboxylate rhodamine, isomerically pure 6-carboxylate rhodamine, and a mixture of 5- and 6-carboxylate rhodamine. isomerically pure 5-carboxylate rhodamine, isomerically pure 6-carboxylate rhodamine, and a mixture of 5- and 6-carboxylate rhodamine.
Claim 12
Independent Claim
442 words
A method of determining the nucleotide sequence of a single stranded DNA, the method comprising the steps of: synthesizing a first, a second, a third, and a fourth fluorescently labeled primer having a base sequence such that (1) the first, second, third, and fourth fluorescently labeled primers each are labeled with a different fluorescent dye, (2) at least one of the different fluorescent dyes is an isomerically pure 6-carboxylrhodamine or an isomerically pure 5-carboxylrhodamine, and (3) the different fluorescent dyes are spectrally resolvable and electrophoretically compatible; inserting the single stranded DNA into a single stranded plasmid adjacent to a complementary region to the base sequence of said primers, such that said primers are capable of priming the synthesis of a complementary oligonucleotide to the inserted single stranded DNA by a DNA polymerase; replicating the single stranded plasmid containing the single stranded DNA; separating the replicated single stranded plasmids into a first portion, a second portion, a third portion, and a fourth portion; annealing the first fluorescently labeled primer to the complementary region of the single stranded plasmids of the first portion and reacting the first fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxyadenosine to form a first set of double stranded DNAs; annealing the second fluorescently labeled primer to the complementary region of the single stranded plasmids of the second portion and reacting the second fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxyguanosine to form a second set of double stranded DNAs; annealing the third fluorescently labeled primer to the complementary region of the single stranded plasmids of the third portion and reacting the third fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxycytidine to form a third set of double stranded DNAs; annealing the fourth fluorescently labeled primer to the complementary region of the single stranded plasmids of the fourth portion and reacting the fourth fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxythymidine to form a fourth set of double stranded DNAs; forming a mixture of the double stranded oligonucleotides from the first, second, third, and fourth sets of double stranded DNAs; melting the double stranded DNAs of the mixture of the first, second, third, and fourth sets of double stranded DNAs to form single stranded oligonucleotides; and separating according to size the single stranded oligonucleotides in the mixture by gel electrophoresis.
Claim 13
Independent Claim
442 words
A method of determining the nucleotide sequence of a single stranded DNA, the method comprising the steps of: synthesizing a first, a second, a third, and a fourth fluorescently labeled primer having a base sequence such that (1) the first, second, third, and fourth fluorescently labeled primers each are labeled with a different fluorescent dye, (2) at least one of the different fluorescent dyes is an isomerically pure 6-carboxylrhodamine or an isomerically pure 5-carboxylrhodamine, and (3) the different fluorescent dyes are spectrally resolvable and electrophoretically compatible; inserting the single stranded DNA into a single stranded plasmid adjacent to a complementary region to the base sequence of said primers, such that said primers are capable of priming the synthesis of a complementary oligonucleotide to the inserted single stranded DNA by a DNA polymerase; replicating the single stranded plasmid containing the single stranded DNA; separating the replicated single stranded plasmids into a first portion, a second portion, a third portion, and a fourth portion; annealing the first fluorescently labeled primer to the complementary region of the single stranded plasmids of the first portion and reacting the first fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxyadenosine to form a first set of double stranded DNAs; annealing the second fluorescently labeled primer to the complementary region of the single stranded plasmids of the second portion and reacting the second fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxyguanosine to form a second set of double stranded DNAs; annealing the third fluorescently labeled primer to the complementary region of the single stranded plasmids of the third portion and reacting the third fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxycytidine to form a third set of double stranded DNAs; annealing the fourth fluorescently labeled primer to the complementary region of the single stranded plasmids of the fourth portion and reacting the fourth fluorescently labeled primer and single stranded plasmid with a DNA polymerase in the presence of triphosphates of adenosine, guanosine, cytidine, thymidine, and dideoxythymidine to form a fourth set of double stranded DNAs; forming a mixture of the double stranded oligonucleotides from the first, second, third, and fourth sets of double stranded DNAs; melting the double stranded DNAs of the mixture of the first, second, third, and fourth sets of double stranded DNAs to form single stranded oligonucleotides; and separating according to size the single stranded oligonucleotides in the mixture by gel electrophoresis.
References Cited (Backward Citations)
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