Method for nucleotide detection

Patent metadata

Field	Value
Publication number	US-9856529-B2
Application number	US-201514729376-A
Country	US
Kind code	B2
Filing date	Jun 3, 2015
Priority date	Jan 31, 2011
Publication date	Jan 2, 2018
Grant date	Jan 2, 2018

Abstract

A method of inhibiting light-induced degradation of nucleic acids includes irradiating a portion of the nucleic acids in the presence of a detection solution comprising a polyphenolic compound. A method of detecting a nucleic acid having a fluorescent tag includes irradiating at least a portion of the nucleic acid with light of a suitable wavelength to induce a fluorescence emission and detecting the fluorescence emission. Optionally, the polyphenolic compound is gallic acid, a lower alkyl ester thereof, or mixtures thereof. A kit includes one or more nucleotides, an enzyme capable of catalyzing incorporation of the nucleotides into a nucleic acid strand and a polyphenolic compound suitable for preparing a detection solution.

First claim

What is claimed is:

1. A method of detecting a nucleic acid having a fluorescent tag comprising:
a) irradiating at least a portion of said nucleic acid with light, wherein said light comprises a suitable wavelength to induce a fluorescence emission;
b) detecting said fluorescence emission; and
c) repeating steps a) and b);
wherein said irradiating step is carried out in the presence of a detection solution comprising gallic acid, a lower alkyl ester thereof, or mixtures thereof, and further comprising urea, said detection solution inhibiting light-induced degradation of said nucleic acid.

2. The method of claim 1 , wherein said nucleic acid is in an array of nucleic acids attached to a support.

3. The method of claim 1 , further comprising adding a fluorescently tagged nucleotide to said nucleic acid.

4. The method of claim 1 , further comprising replacing a solution with said detection solution prior to the irradiation step.

5. The method of claim 2 , wherein inhibiting light-induced degradation to said nucleic acid comprises reducing the cleavage of a nucleic acid member from said array.

6. The method of claim 1 , wherein said gallic acid, said lower alkyl ester thereof, or said mixtures thereof is present in a concentration ranging from between about 10 mM to about 200 mM.

7. The method of claim 1 , further comprising one or more compound(s), wherein said one or more compound(s) further enhances preservation of the integrity of said nucleic acid.

8. The method of claim 7 , wherein said one or more compound(s) is selected from ascorbic acid or salt thereof, and isoascorbic acid or salt thereof.

9. The method of claim 1 , comprising at least 50 cycles repeating step c.

10. The method of claim 1 , comprising repeating said adding and detection steps for a number of cycles in a range from between about 100 cycles to about 1,000 cycles.

11. The method of claim 1 , wherein the presence of said detection solution reduces a detection error rate by greater than 20% relative to a control.

12. The method of claim 11 , wherein one or more compound(s) selected from ascorbic acid or salt thereof, and isoascorbic acid or salt thereof further reduce(s) said detection error rate.

13. The method of claim 1 , wherein the presence of said detection solution reduces a detection error rate by greater than 40% relative to a control.

14. The method of claim 13 , wherein one or more compound(s) selected from ascorbic acid or salt thereof, and isoascorbic acid or salt thereof further reduce(s) said detection error rate.

15. The method of claim 1 , wherein the presence of said detection solution reduces a detection error rate by greater than 50% relative to a control.

16. The method of claim 15 , wherein one or more compound(s) selected from ascorbic acid or salt thereof, and isoascorbic acid or salt thereof further reduce(s) said detection error rate.

17. The method of claim 1 , wherein said irradiation step is conducted in a range from about 360 nm to about 700 nm.

18. The method of claim 1 , wherein said irradiation step is conducted with a light source having power in a range between about 5 to about 500 milliwatts.

19. The method of claim 1 , wherein said irradiation step is conducted for a time period of about 0.1 seconds to about 10 minutes.

20. The method of claim 2 , wherein adding an additional fluorescently tagged nucleotide comprises using a polymerase to add a single nucleotide.

Assignees

• Illumina Inc

Inventors

• Klausing Kay
• Shen Min-Jui Richard
• Moore John
• Smith Vincent Peter
• Hall Kevin

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2527/127

  the enzyme inhibitor or activator used · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6874 Primary

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