Method of nucleotide detection

Patent metadata

Field	Value
Publication number	US-9970055-B2
Application number	US-201615050337-A
Country	US
Kind code	B2
Filing date	Feb 22, 2016
Priority date	Dec 13, 2004
Publication date	May 15, 2018
Grant date	May 15, 2018

Abstract

The invention relates to an additive which can be added to buffers used in nucleotide detection processes and improved methods of nucleic acid sequencing using this additive. In particular the invention relates to use of the additive to improve the efficiency of fluorescence-based multiple cycle nucleic acid sequencing reactions.

First claim

The invention claimed is:

1. A kit for sequencing a nucleic acid template, the kit comprising:
a plurality of fluorescently labelled nucleotides;
an enzyme capable of catalyzing incorporation of the fluorescently labelled nucleotides into a nucleic acid strand complementary to the nucleic acid template to be sequenced; and
a buffer comprising ascorbic acid or a salt thereof, or a supply of ascorbic acid or a salt thereof suitable for preparing the buffer.

2. The kit of claim 1 , wherein the salt of ascorbic acid is sodium ascorbate.

3. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration of at least 10 mM.

4. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration of at least 20 mM.

5. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration of up to 50 mM.

6. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration of up to 100 mM.

7. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration ranging from 10 to 100 mM.

8. The kit of claim 1 , wherein the ascorbic acid or salt thereof is present in the buffer at a concentration ranging from 20 to 50 mM.

9. The kit of claim 1 , wherein the buffer has a pH of about 5 to about 9.

10. The kit of claim 1 , wherein the buffer has a pH of about 5.5 to about 8.6.

11. The kit of claim 1 , wherein the buffer has a pH of about 7.

12. The kit of claim 1 , wherein the fluorescently labelled nucleotides are dNTPs.

13. The kit of claim 1 , further comprising an enzyme cofactor required for incorporation of the fluorescently labelled nucleotides into a growing nucleic acid strand.

14. The kit of claim 1 , wherein the buffer further comprises an enzyme, an enzyme cofactor, an oligonucleotide primer, dNTPs, or a combination thereof.

15. The kit of claim 1 , wherein the buffer further comprises an additional antioxidant.

16. The kit of claim 1 , wherein the fluorescent label is attached to the nucleotides by a linker comprising a disulfide.

17. The kit of claim 1 , wherein the fluorescent label is attached to the nucleotides by a linker comprising an azide.

18. The kit of claim 17 , wherein the azide containing linker is cleavable by a phosphine.

Assignees

• Illumina Cambridge Ltd

Inventors

• Fedurco Milan
• Romieu Anthony
• Turcatti Gerardo

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2523/107

  Chemical cleaving agents · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2563/107

  fluorescence · CPC title