Method for nucleotide detection

• US 9,856,529 B2
• Filed: 06/03/2015
• Issued: 01/02/2018
• Est. Priority Date: 01/31/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Klausing, Kay, Shen, Min-Jui Richard, Moore, John, Smith, Vincent Peter, Hall, Kevin
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US14/729,376
• Publication Number:
  US 20150259737A1
• Time in Patent Office: 944 Days
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes

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.

Claims

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.