Method for nucleotide detection

Patent metadata

Field	Value
Publication number	US-9115353-B2
Application number	US-201113018255-A
Country	US
Kind code	B2
Filing date	Jan 31, 2011
Priority date	Jan 31, 2011
Publication date	Aug 25, 2015
Grant date	Aug 25, 2015

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 inhibiting light-induced degradation of nucleic acids during a detection step, the method comprising:
introducing an incorporation solution into a flow cell, said incorporation solution comprising polymerase and a fluorescently tagged nucleotide, said flow cell comprising an array of nucleic acids attached to a support;
replacing the incorporation solution with a detection solution comprising gallic acid, a lower alkyl ester thereof, or mixtures thereof, and further comprising urea,
irradiating a portion of said nucleic acids in the presence of said detection solution, wherein said detection solution reduces the amount of light-induced degradation of said nucleic acids.

2. 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.

3. The method of claim 1 , further comprising adding an additional fluorescently tagged nucleotide to said array and repeating said detection step in a cycle.

4. The method of claim 3 , comprising repeating said adding and detection steps for at least 50, 75, or 100 cycles.

5. The method of claim 3 , 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.

6. 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.

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

8. 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.

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

10. The method of claim 1 , wherein said array comprises a primer template.

11. A method of detecting a nucleic acid having a fluorescent tag comprising:
a) introducing an incorporation solution into a flow cell, said incorporation solution comprising polymerase and a fluorescently tagged nucleotide, said flow cell comprising an array of nucleic acids attached to a support, to add a fluorescently tagged nucleotide to said nucleic acid;
b) replacing the incorporation solution with a detection solution;
c) irradiating at least a portion of said nucleic acid with light in the presence of the detection solution, wherein said light comprises a suitable wavelength to induce a fluorescence emission;
d) detecting said fluorescence emission; and
e) repeating steps a) through d);
wherein the detection solution comprises 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.

12. The method of claim 11 , 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.

13. The method of claim 11 , comprising at least 50, 75, or 100 cycles repeating step e.

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

15. The method of claim 11 , wherein said irradiation step is conducted in a range from about 360 nm to about 700 nm.

16. The method of claim 11 , wherein said irradiation step is conducted with a light source having power in a range between about 5 to about 500 milliwatts.

17. The method of claim 11 , wherein said irradiation step is conducted for a time period of about 0.1 seconds to about 10 minutes.

18. The method of claim 11 , wherein step a) comprises using a polymerase to add a single nucleotide.

19. The method of claim 11 , wherein said array comprises a primer template.

Assignees

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

Inventors

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

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6874 Primary

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

• C12N15/1003 Primary

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

• C40B20/04 Primary

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title