Spectrally resolvable rhodamine dyes for nucleic acid sequence determination

• US 5,366,860 A
• Filed: 09/29/1989
• Issued: 11/22/1994
• Est. Priority Date: 09/29/1989
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Eadie, J. Scott, Lee, Linda G., Menchen, Steven M., Woo, Sam L., Connell, Charles R., Fung, Steven, Chakerian, Vergine, Hershey, N. Davis, Bergot, B. John
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): Hendricks, Keith D.
• Application Number:
  US07/415,050
• Time in Patent Office: 1,880 Days
• Field of Search: 435/6
• US Class Current: 435/6.12
• 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: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

A spectrally resolvable set of rhodamine dyes are provided for use in the chain termination method of nucleic acid sequencing. A different rhodamine dye from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110 is attached to the base of each of the dideoxynucleotides used in the sequencing method by way of an alkynylamino linker. Preferably, the labeled dideoxynucleotides are incorporated into the growing DNA chains by Taq DNA polymerase.

Claims

1. A method of distinguishing polynucleotides having different 3'"'"'-terminal dideoxynucleotides in the chain termination method of DNA sequencing, the method comprising the steps of:

forming a mixture of a first, a second, a third, and a fourth class of polynucleotides, each polynucleotide in the first class having a 3'"'"'-terminal dideoxyadenosine and being labeled with a first dye selected from the going consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;

each polynucleotide in the second class having a 3'"'"'-terminal dideoxycytidine and being labeled with a second dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;

each polynucleotide in the third class having a 3'"'"'-terminal dideoxyguanosine and being labeled with a third dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110; and

each polynucleotide in the fourth class having a 3'"'"'-terminal dideoxythymidine and being labeled with a fourth dye selected from the group consisting of tetramethylrhodamine, rhodamine X, rhodamine 6G, and rhodamine 110;

wherein each class of polynucleotide in the mixture is labeled with a different dye;

electrophoretically separating on a gel the polynucleotides in the mixture so that bands of similarly sized polynucleotides are formed;

illuminating with an illumination beam the bands on the gel, the illumination beam being capable of causing the dyes to fluoresce; and

identifying the classes of the polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.

2. The method of claim 1 wherein said 3'"'"'-terminal dideoxyadenosine is 2'"'"',3'"'"'-dideoxy-7-deazaadenosine and said first dye is attached by way of a linking group to a 7 carbon atom thereof;

said 3'"'"'-terminal dideoxycytidine is 2'"'"',3'"'"'-dideoxycytidine and said second dye is attached by way of a linking group to a 5 carbon atom thereof;

said 3'"'"'-terminal dideoxyguanosine is selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine and said third dye is attached by way of a linking group to a 7 carbon atom thereof; and

said 3'"'"'-terminal dideoxythymidine is 2'"'"',3'"'"'-dideoxyuridine and said fourth dye is attached by way of a linking group to a 5 carbon atom thereof.

3. The method of claim 2 wherein said linking group is 3-carboxyamino-1-propynyl and links said 7 carbon atoms of said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine and said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine to a 5 or 6 carbon atom of said first and third dyes, respectively, and links said 5 carbon atoms of said dideoxycytidine and said dideoxyuridine to a 5 or 6 carbon atom of said second and fourth dyes, respectively.

4. The method of claim 3 further including the step of extending from a primer a plurality of polynucleotides by means of a DNA polymerase in the presence of dideoxyadenosine triphosphate, dideoxycytidine triphosphate, a dideoxyguanosine triphosphate, and a dideoxythymidine triphosphate to form said first, second, third, and fourth classes of polynucleotides.

5. The method of claim 4 wherein said DNA polymerase is selected from the group consisting of Taq DNA polymerase and T7 DNA polymerase.

6. The method of claim 5 wherein said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine is attached to said 5 carbon atom of rhodamine 6G, said 2'"'"',3'"'"'-dideoxycytidine is attached to said 6 carbon atom of rhodamine X, said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine is attached to said 5 carbon atom of rhodamine 110, and said 2'"'"',3'"'"'-dideoxyuridine is attached to said 6 carbon atom of tetramethylrhodamine.

7. The method of claim 5 wherein said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine is attached to said 6 carbon atom of rhodamine X, said 2'"'"',3'"'"'-dideoxycytidine is attached to said 5 carbon atom of rhodamine 6G, said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine is attached to said 5 carbon atom of rhodamine 110, and said 2'"'"',3'"'"'-dideoxyuridine is attached to said 6 carbon atom of tetramethylrhodamine.

8. The method of claim 5 wherein said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine is attached to said 6 carbon atom of rhodamine X, said 2'"'"',3'"'"'-dideoxycytidine is attached to said 5 carbon atom of rhodamine 110, said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine is attached to said 6 carbon atom of tetramethylrhodamine, and said 2'"'"',3'"'"'-dideoxyuridine is attached to said 5 carbon atom of rhodamine 6G.

9. The method of claim 5 wherein said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine is attached to said 5 carbon atom of rhodamine 6G, said 2'"'"',3'"'"'-dideoxycytidine is attached to said 5 carbon atom of rhodamine 110, said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine is attached to said 6 carbon atom of rhodamine X, and said 2'"'"',3'"'"'-dideoxyuridine is attached to said 6 carbon atom of tetramethylrhodamine.

10. The method of claim 5 wherein said 2'"'"',3'"'"'-dideoxy-7-deazaadenosine is attached to said 6 carbon atom of rhodamine X, said 2'"'"',3'"'"'-dideoxycytidine is attached to said 6 carbon atom of tetramethylrhodamine, said dideoxyguanosine selected from the group consisting of 2'"'"',3'"'"'-dideoxy-7-deazaguanosine and 2'"'"',3'"'"'-dideoxy-7-deazainosine is attached to said 5 carbon atom of rhodamine 6G, and said 2'"'"',3'"'"'-dideoxyuridine is attached to said 5 carbon atom of rhodamine 110.