# US 7,407,747 B2 · Method for drying dye-terminator sequencing reagents

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- Number: US 7,407,747 B2
- Document ID: US7407747B2
- Status: Granted
- Kind: B2
- Application number: 10/272,450
- Filed: 2002-10-15
- Published: 2004-04-15
- Granted: 2008-08-05
- Earliest claimed: 2002-10-15
- Assignees: Applera Corporation
- Inventors: Kathleen M. Perry; Robert E. Ruhfel; Sandra L. Spurgeon; Peter Ma
- Web page: https://patentgrep.com/patent/US7407747B2

## CPC

- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6869: Methods for sequencing
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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

### Claim 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.

### Claim 2 (depends on 1)

The composition according to claim 1 further comprising a cryoprotectant.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 2)

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

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 5)

The method of claim 5 wherein the cryoprotectant is trehalose.

### Claim 7 (depends on 5)

The method of claim 5 wherein the cryoprotectant is sucrose.

### Claim 8 (depends on 5)

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.

### Claim 9 (depends on 5)

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

### Claim 10 (depends on 9)

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.

### Claim 11 (depends on 10)

The method of claim 10 wherein the dye has the structure
where R21 is a C1-5 alkyl; Z1 is NH, sulfur, or oxygen; R22 is an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system; R29 is C1-C5 alkyl; and Z2 is NH, sulfur, or oxygen.

### Claim 12 (depends on 9)

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

### Claim 13 (depends on 12)

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.

### Claim 14 (depends on 9)

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

### Claim 15 (depends on 14)

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.