Method for drying dye-terminator sequencing reagents

• US 7,407,747 B2
• Filed: 10/15/2002
• Issued: 08/05/2008
• Est. Priority Date: 10/15/2002
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Ma, Peter, Spurgeon, Sandra L., Ruhfel, Robert E., Perry, Kathleen M.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/272,450
• Publication Number:
  US 20040072167A1
• Time in Patent Office: 2,121 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/25.3, 536/26.6
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

A dry composition comprising a nucleotide linked to a fluorescent dye is provided. A method of making a dry composition comprising the step of drying a solution comprising a nucleotide linked to a fluorescent dye is provided. A method is provided for performing a reaction on a nucleic acid substrate comprising: (a) providing a vessel containing a dried composition comprising a nucleotide linked to a fluorescent dye, and (b) adding to the vessel an aqueous solution and the nucleic acid substrate. A kit is provided for determining the sequence of a nucleic acid, the kit comprising a dry composition comprising a nucleotide linked to a fluorescent dye.

Claims

1. A dry composition comprising a mononucleotide linked to a fluorescent dye, wherein the composition comprises a thermostable nucleic acid polymerase, wherein the thermostable nucleic acid polymerase is Taq DNA polymerase, wherein the dry composition is substantially free of glycerol, and wherein the ratio of glycerol to Taq DNA polymerase is less than 1 ug of glycerol per unit of Taq DNA polymerase.

2. The composition according to claim 1 further comprising a cryoprotectant.

3. The composition according to claim 2 wherein the cryoprotectant is trehalose.

4. The composition according to claim 2 wherein the cryoprotectant is sucrose.

5. A method of making the composition of claim 1 comprising the step of drying a solution comprising the mononucleotide linked to the fluorescent dye, the Taq DNA polymerase, and a cryoprotectant.

6. The method of claim 5 wherein the cryoprotectant is trehalose.

7. The method of claim 5 wherein the cryoprotectant is sucrose.

8. The method of claim 5 wherein the temperature of the solution during at least a portion of the drying step is less than 0°

C.

9. The method of claim 5 wherein the dye is an energy transfer dye comprising a donor connected to an acceptor by a linker.

10. The method of claim 9 wherein the linker comprises an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system.

11. The method of claim 10 wherein the dye has the structure

12. The method of claim 9 wherein the donor is a fluorescein, rhodamine, or asymmetric benzoxanthene dye.

13. The method of claim 12 wherein the donor is a carboxyfluorescein;

4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichiororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

14. The method of claim 9 wherein the acceptor is a xanthene, cyanine, phthalocyanine, or squaraine dye.

15. The method of claim 14 wherein the acceptor is a 4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichiororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.