Composition for stabilization of labelled nucleoside triphosphates and methods for using same

• US 5,808,043 A
• Filed: 01/18/1995
• Issued: 09/15/1998
• Est. Priority Date: 01/18/1995
• Status: Expired due to Fees

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Pharmacia Biotech Inc. (General Electric Company)
• Inventors: Duthie, R. Scott, Freeman, Mark E., Stirchak, Eugene P., Burazin, Lawrence J., Brush, Charles K.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/374,456
• Time in Patent Office: 1,336 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.32, 536/25.5, 536/26.26, 536/26.7, 536/26.8, 536/26.41, 536/26.42, 435/6, 435/7.91, 435/89
• US Class Current: 536/25.32
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids

Abstract

A preparation of a labelled nucleotide comprising at least one compound having a Mg²⁺ association constant between 1×10^-11 to 1×10^-2, inclusive. The compound is preferably selected from the group consisting of citrate, isocitrate, phosphate, EGTA, EDTA, and CDTA. The concentration of the compound is preferably at least 5 mM.

Claims

1. A preparation comprising:

a labelled nucleotide; and

at least one compound having a Mg²⁺ association constant between 1×

10^-11 to 1×

10^-2, inclusive, wherein the concentration of the compound is at least 5 mM.

2. The preparation of claim 1, the labelled nucleotide having the formula PM-SM-BASE-SIG wherein PM is a phosphate moiety, SM is a sugar moiety, BASE is a pyrimidine, purine or 7-deazapurine moiety, PM being attached at the 3'"'"' or the 5'"'"' position of SM when the nucleotide is a deoxyribonucleotide and at the 2'"'"', 3'"'"' or 5'"'"' position when the nucleotide is a ribonucleotide, BASE being attached to the 1'"'"' position of SM from the N¹ position when BASE is a pyrimidine or the N⁹ position when BASE is a purine or a 7-deazapurine, and SIG is covalently attached to BASE and wherein SIG represents a detectable moiety.

3. The preparation of claim 2, wherein the at least one compound is selected from the group consisting of citrate, isocitrate, phosphate, EGTA, EDTA, and CDTA.

4. The preparation of claim 3, wherein SIG is selected from the group consisting of fluorophores, and chromophores.

5. The preparation of claim 3, wherein SIG is selected from the group consisting of fluoresceins, carbocyanines, and rhodamines.

6. The preparation of claim 5, wherein SIG is a fluorescein.

7. The preparation of claim 6, wherein the labelled nucleotide is fluorescein-labelled dATP.

8. The preparation of claim 5, wherein SIG is a carbocyanine.

9. The preparation of claim 8, wherein the labelled nucleotide is carbocyanine-labelled dATP.

10. The preparation of claim 1, wherein the labelled nucleotide is covalently attached to at least one nucleoside.

11. The preparation of claim 1, wherein the compound is a carboxylic acid.

15. In a polymerization process catalyzed by DNA polymerase, the improvement comprising using the preparation of claim 1 as a substrate.

16. In a polymerization process catalyzed by RNA polymerase, the improvement comprising using the preparation of claim 1 as a substrate.

17. In a polymerization process catalyzed by reverse transcriptase, the improvement comprising using the preparation of claim 1 as a substrate.

12. A method of stabilizing a labelled nucleotide comprising the step of:

adding to the labelled nucleotide at least one compound having a Mg²⁺ association constant from 1×

10^-11 to 1×

10^-2, wherein the final concentration of the compound is at least 5 mM.

13. The method of claim 12, comprising the further step of first removing pyrogens from the constituents.

14. The method of claim 13, wherein the removing step is accomplished by filtering.