Method of detecting electrophoretically separated oligonucleotides

Claim 1

A method for detecting up to four classes of oligonucleotides separated by gel electrophoresis, the method comprising the steps of: labelling each oligonucleotide within each class with one or more dyes selected from the same one of either a first set of dyes, a second set of dyes, a third set of dyes, or a fourth set of dyes to form dye-oligonucleotide conjugates from each class such that oligonucleotides from the same class are labelled with dyes from the same set and oligonucleotides from different classes are labelled with dyes from different sets, the first set consisting of dyes defined by the formula: wherein A is a linking functionality and B is an acidic anionic group, the second set consisting of dyes defined by the formula: wherein A and B are defined as above, the third set consisting of dyes defined by the formula: wherein A and B are defined as above, and the fourth set consisting of dyes defined by the formula: wherein A' is a linking functionality or an acidic anionic group and B' is a linking functionality whenever A' is an acidic anionic group, and B' is an acidic anionic group whenever A' is a linking functionality; forming a mixture of the dye-oligonucleotide conjugates from more than one class; electrophoretically separating the dye-oligonucleotide conjugates on a gel; illuninating the dyes of similarly separated dye-oligonucleotide conjugates; and identifying the class of similarly separated dye-oligonucleotide conjugates by the fluorescence or absorption spectrum of the dyes of the dye-oligonucleotide conjugates.

Claim 2

A method for detecting up to four classes of oligonucleotides separated by gel electrophoresis, the method comprising the steps of: labelling each oligonucleotide within each class with one or more dyes selected from the same one of either a first set of dyes, a second set of dyes, a third set of dyes, or a fourth set of dyes to form dye-oligonucleotide conjugates from each class such that oligonucleotides from the same class are labelled with dyes from the same set and oligonucleotides from different classes are labelled with dyes from different sets, the first set consisting of dyes defined by the formula: wherein A is a linking functionality and B is an acidic anionic group, the second set consisting of dyes defined by the formula: wherein A and B are defined as above, the third set consisting of dyes defined by the formula: wherein A and B are defined as above, and the fourth set consisting of dyes defined by the formula: wherein A' is a linking functionality or an acidic anionic group and B' is a linking functionality whenever A' is an acidic anionic group, and B' is an acidic anionic group whenever A' is a linking functionality; forming a mixture of the dye-oligonucleotide conjugates from more than one class; electrophoretically separating the dye-oligonucleotide conjugates on a gel; illuninating the dyes of similarly separated dye-oligonucleotide conjugates; and identifying the class of similarly separated dye-oligonucleotide conjugates by the fluorescence or absorption spectrum of the dyes of the dye-oligonucleotide conjugates.

Claim 3

A method of distinguishing oligonucleotides having different terminal dideoxyribonucleotides in the enzymatic method of DNA sequencing, the method comprising the steps of: linking a first dye to the 5' termini of oligonucleotides of a predetermined sequence to form a first plurality of labelled primers, the first dye being selected from the group defined by the formula: wherein A is a linking functionality and B is an acidic anionic group; linking a second dye to the 5' termini of oligonucleotides of the predetermined sequence to form a second plurality of labelled primers, the second dye being selected from the group defined by the formula: wherein A and B are defined as above; linking a third dye to the 5' termini of oligonucleotides of the predetermined sequence to form a third plurality of labelled primers, the third dye being selected from the group defined by the formula: wherein A and B are defined as above; linking a fourth dye to the 5' termini of oligonucleotides of the predetermined sequence to form a fourth plurality of labelled primers, the fourth dye being selected from the group defined by the formula: wherein A' is a linking functionality or an acidic anionic group, and B' is a linking functionality whenever A' is an acidic anionic group and B' is an acidic anionic group whenever A' is a linking functionality; using the first, second, third and fourth pluralities of labelled primers to separately generate labelled oligonucleotides in accordance with an enzymatic DNA sequencing procedure such that a one-to-one correspondence is established between the pluralities of labelled primers and the kind of dideoxyribonucleotide attached to the 3' terminus of the labelled oligonucleotide; mixing the labelled oligonucleotides; electrophoretically separating the labelled oligonucleotides on a gel according to size; illuminating similarly separated labelled oligonucleotides; and identifying the kind of dideoxyribonucleotide attached to the 3' terminus of similarly separated labelled oligonucleotides by relating the respective fluorescence or absorption spectrum of the similarly separated labelled oligonucleotides to the attached dideoxyribonucleotide.

Claim 4

A method of distinguishing oligonucleotides having different terminal dideoxyribonucleotides in the enzymatic method of DNA sequencing, the method comprising the steps of: linking a first dye to the 5' termini of oligonucleotides of a predetermined sequence to form a first plurality of labelled primers, the first dye being selected from the group defined by the formula: wherein A is a linking functionality and B is an acidic anionic group; linking a second dye to the 5' termini of oligonucleotides of the predetermined sequence to form a second plurality of labelled primers, the second dye being selected from the group defined by the formula: wherein A and B are defined as above; linking a third dye to the 5' termini of oligonucleotides of the predetermined sequence to form a third plurality of labelled primers, the third dye being selected from the group defined by the formula: wherein A and B are defined as above; linking a fourth dye to the 5' termini of oligonucleotides of the predetermined sequence to form a fourth plurality of labelled primers, the fourth dye being selected from the group defined by the formula: wherein A' is a linking functionality or an acidic anionic group, and B' is a linking functionality whenever A' is an acidic anionic group and B' is an acidic anionic group whenever A' is a linking functionality; using the first, second, third and fourth pluralities of labelled primers to separately generate labelled oligonucleotides in accordance with an enzymatic DNA sequencing procedure such that a one-to-one correspondence is established between the pluralities of labelled primers and the kind of dideoxyribonucleotide attached to the 3' terminus of the labelled oligonucleotide; mixing the labelled oligonucleotides; electrophoretically separating the labelled oligonucleotides on a gel according to size; illuminating similarly separated labelled oligonucleotides; and identifying the kind of dideoxyribonucleotide attached to the 3' terminus of similarly separated labelled oligonucleotides by relating the respective fluorescence or absorption spectrum of the similarly separated labelled oligonucleotides to the attached dideoxyribonucleotide.

Claim 6

A method according to any one of claims 1 to 3, wherein the acidic anionic group of the substituents A', B and B' is carboxlic acid or sulfonic acid.

Claim 8

A method according to any one of claims 1 to 5, wherein the first set of dyes consists of, or the first dye is selected from fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, fluorescein-5-succinimidylcarboxylate, and fluorescein-6-succinimidylcarboxylate; the second set of dyes consists of, or the second dye is selected from 2',7'-dimethoxy-4',5'-dichlorofluorescein-5-succinimidylcarboxylate, and 2',7'-dimethoxy-4',5'-di- chlorofluorescein-6-succinimidylcarboxylate; the third set of dyes consists of, or the third dye is selected from tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, tetramethylrhodamine-5-succinimidylcarboxylate, and tetramethylrhodamine-6-succinimidylcarboxylate; and the fourth set of dyes consists of, or the fourth dye is selected from Texas Red, rhodamine X-5-isothiocyanate, rhodamine X-6-isothiocyanate, rhodamine X-5-succinimidylcarboxylate, and rhodamine X-6-succinimidylcarboxylate.

Claim 9

A method according to any one of claims 3 to 6, wherein the step of attaching the complementary functionality includes reacting a 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkane with the oligonucleotides.

Claim 11

A method according to any one of claims 1 to 8, wherein the gel is polyacrylamide gel having a concentration of 2 to 25 percent.

Claim 12

A method according to any one of claims 1 to 9, wherein the step of illuminating includes illuminating the dyes with 514 nm light.

Claim 13

A method according to any one of claims 1 to 9, wherein the step of illuminating includes illuminating the dyes with 488 nm light.

Claim 14

A method according to any one of claims 1 to 11, wherein the step of identifying includes identifying similarly separated dye-oligonucleotide conjugates or labelled oligonucleotides by the fluorescence spectrum of the dyes of the dye-oligonucleotide conjugates, or labelled primers of the labelled oligonucleotides.