Stabilizer of dye sequencing products

• US 6,232,076 B1
• Filed: 02/04/2000
• Issued: 05/15/2001
• Est. Priority Date: 02/04/2000
• Status: Expired due to Fees

• Current Assignee: Genaissance Pharmaceuticals Incorporated (Abbvie Incorporated)
• Original Assignee: Genaissance Pharmaceuticals Incorporated (Abbvie Incorporated)
• Inventors: Schulz, Vincent P.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/498,270
• Time in Patent Office: 466 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Methods for reducing degradation of polymerase extension products in the presence of formamide, where the polymerase extension products comprise a nonradioactive detection moiety, are disclosed. The methods comprise adding a base, a buffer or a reducing agent to the polymerase extension products. Sequencing methods which generate polymerase extension products comprising a nonradioactive detection moiety are also disclosed, wherein the degradation of the extension products is reduced or eliminated by the addition of a base, a buffer, or a reducing agent. Compositions for reducing degradation of the above polymerase extension products are also disclosed, where the compositions comprise a base, a buffer, or a reducing agent. Kits for performing sequencing methods are also disclosed, wherein the degradation of polymerase extension products is reduced or eliminated. The kits comprise a base, a buffer, or a reducing agent, and instructions.

Claims

1. A method of reducing degradation of fluorescent dye-labeled polymerase extension products, comprising adding to the extension products formamide and at least one additional compound selected from the group consisting of a base, a buffer and a reducing agent, wherein the additional compound is not alkaline ethylenediaminetetraacetic acid (EDTA), and wherein less degradation of polymerase extension products occurs in the presence of formamide and the additional compound a in the presence of formamide without the additional compound.

2. The method of claim 1, wherein the at least one compound is dithiothreitol (DTT).

3. The method of claim 2, wherein the method further comprises adding NaHCO₃.

4. The method of claim 1, wherein the fluorescent dye is a fluorescent energy transfer dye.

5. The method of claim 4, wherein the fluorescent energy transfer dye is 5CFB-dR110-2.

6. The method of claim 4, wherein the at least one compound is DTT and the method further comprises adding NaHCO₃.

7. The method of claim 4, wherein the polymerase extension products terminate in four different dideoxyribonucleotides and at least one of the dideoxyribonucleotides is labeled with the fluorescent energy transfer dye.

8. The method of claim 7, wherein the at least one labeled dideoxyribonucleotide is ddG-EO-5CFB-dR110-2.

9. The method of claim 8, wherein addition of the compound prevents excessive fluorescence at about 530 nm, wherein the excessive fluorescence migrates in polyacrylamide gel electrophoresis or capillary electrophoresis similar to a polymerase extension product which is about 90 bases long.

19. The method of claim 3, wherein the DTT is added at 5 mM and the NaHCO₃ is added at 0.3 mM.

20. The method of claim 4, wherein the DTT is added at 5 mM and the NaHCO₃ is added at 0.3 mM.

10. A method for determining the sequence of a DNA template which comprises

(a) preparing a sequencing reaction mixture comprising

(i) the DNA template;

(ii) a primer which is a complementary to a 3′

region of the DNA template;

(iii) unlabeled deoxyribonucleoside triphosphates;

(iv) at least one dideoxyribonucleoside triphosphate;

(v) a DNA polymerase;

(vi) a substance labeled with a fluorescent dye, wherein the substance is a deoxyribonucleoside triphosphate, the primer, or the at least one dideoxyribonucleoside triphosphate; and

(vii) an aqueous buffer

(b) treating the sequencing reaction mixture under conditions and for a time sufficient to synthesize a series of polymerase extension products of different lengths, wherein each of the polymerase extension products comprises a sequence of nucleotides complementary to at least part of the DNA template and wherein at least one of the nucleotides in each extension product comprises the fluorescent dye;

(c) mixing the polymerase extension products with a denaturing solution comprising formamide, wherein the denaturing solution further comprises at least one compound that reduces degradation of the nucleotide comprising the fluorescent dye, wherein the at least one compound is not alkaline ethylenediaminetetraacetic acid (EDTA); and

(d) analyzing the denatured polymerase extension products to determine the sequence of nucleotides in the DNA template.

11. The method of claim 10, wherein the at least one compound in the denaturing solution is selected from the group consisting of a base, a buffer, and a reducing agent.

12. The method of claim 11, wherein the at least one compound of step (c) is dithiothreitol (DTT).

13. The method of claim 12, wherein the denaturing solution further comprises NaHCO₃.

14. The method of claim 11, wherein the fluorescent dye is a fluorescent energy transfer dye.

15. The method of claim 14, wherein the fluorescent energy transfer dye is 5CFB-dR110-2.

16. The method of claim 15, wherein the conditions of step (b) are conducive for cycle amplification of the polymerase extension products.

17. The method of claim 16, wherein the labeled nucleotide is a dideoxyribonucleotide.

18. The method of claim 17, wherein the labeled dideoxyribonucleotide is ddG-EO-5CFB-dR110-2.

21. The method of claim 12, wherein the DTT is present at 5 mM and the NaHCO₃ is present at 0.3 mM.