METHOD FOR NUCLEOTIDE DETECTION

• US 20120196758A1
• Filed: 01/31/2011
• Published: 08/02/2012
• Est. Priority Date: 01/31/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Shen, Min-Jui Richard, KLAUSING, Kay, Smith, Vincent, Moore, John, Hall, Kevin
• Granted Patent: US 9,115,353 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 inhibiting light-induced degradation of nucleic acids during a detection step comprising irradiating a portion of said nucleic acids in the presence of a detection solution comprising gallic acid, a lower alkyl ester thereof, or mixtures thereof, wherein said detection solution reduces the amount of light-induced degradation of said nucleic acids.

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

5. 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.

6. The method of claim 1, further comprising one or more compound(s), wherein said one or more compound(s) further reduces the amount of light-induced degradation of said array of nucleic acids.

7. The method of claim 6, wherein said one or more compound(s) is(are) selected from urea, ascorbic acid or salt thereof, and isoascorbic acid or salt thereof.

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

10. The method of claim 8, comprising repeating said adding and detection steps for at least 50, 75, or 100 cycles.

13. The method of claim 8, 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.

16. 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.

17. The method of claim 16, further comprising one or more compounds, wherein said one or more compound are selected from urea, ascorbic acid or salt thereof, and isoascorbic acid or salt thereof, and wherein the one or more compounds further reduces said detection error rate.

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

23. 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.

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

25. The method of claim 2, wherein said array comprises a primer template.

3. -4. (canceled)

9. (canceled)

11. -12. (canceled)

14. -15. (canceled)

18. -21. (canceled)

26. 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, said detection solution inhibiting light-induced degradation of said nucleic acid.

27. The method of claim 26, wherein said nucleic acid is in an array of nucleic acids attached to a support.

28. The method of claim 26, further comprising adding a fluorescently tagged nucleotide to said nucleic acid.

31. The method of claim 26, 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.

32. The method of claim 26, further comprising one or more compound(s), wherein said one or more compound(s) further enhances preservation of the integrity of said nucleic acid.

33. The method of claim 32, wherein said one or more compound(s) is selected from urea, ascorbic acid or salt thereof, and isoascorbic acid or salt thereof.

34. The method of claim 26, comprising at least 50, 75, or 100 cycles repeating step c.

40. The method of claim 26, wherein the presence of said detection solution reduces a detection error rate by greater than 20% relative to a control.

41. The method of claim 40, further comprising one or more compounds, wherein said one or more compound are selected from urea, ascorbic acid or salt thereof, and isoascorbic acid or salt thereof, and wherein the one or more compounds further reduces said detection error rate.

46. The method of claim 26, wherein said irradiation step is conducted in a range from about 360 nm to about 700 nm.

47. The method of claim 26, wherein said irradiation step is conducted with a light source having power in a range between about 5 to about 500 milliwatts.

48. The method of claim 26, wherein said irradiation step is conducted for a time period of about 0.1 seconds to about 10 minutes.

49. The method of claim 27, wherein adding an additional fluorescently tagged nucleotide comprises using a polymerase to add a single nucleotide.

50. The method of claim 27, wherein said array comprises a primer template.

29. -30. (canceled)

35. -39. (canceled)

42. -45. (canceled)