Photodamage mitigation compounds and systems

• US 9,732,382 B2
• Filed: 08/11/2014
• Issued: 08/15/2017
• Est. Priority Date: 08/12/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Weber, Robert, Fedorov, Andrei, Kong, Xiangxu, Lyle, John, Yue, Stephen
• Primary Examiner(s): Hartley, Michael G
• Assistant Examiner(s): LAMBERSKI, JENNIFER A
• Application Number:
  US14/456,590
• Publication Number:
  US 20150079603A1
• Time in Patent Office: 1,100 Days
• Field of Search: None
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2333/91245 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

Compositions, devices, systems and methods for reducing and/or preventing photo-induced damage of one or more reactants in an illuminated analytical reaction by addition of one or more photoprotective compounds to the reaction mixture and allowing the reaction to proceed for a period that is less than a photo-induced damage threshold period.

Claims

1. A method for protecting an enzyme from photo-induced damage in an illuminated reaction, the method comprising:

providing a reaction mixture comprising the enzyme, and a fluorescent or fluorogenic substrate for the enzyme, wherein interaction of the enzyme and the fluorescent or fluorogenic substrate under excitation illumination results in altered activity of the enzyme;

adding a photoprotective agent to the reaction mixture, wherein the photoprotective agent comprises a compound of a formula selected from the group consisting of;

2. The method of claim 1, further comprising the step of monitoring a reaction between the enzyme and the fluorescent or fluorogenic substrate while illuminating the reaction mixture.

3. The method of claim 1, wherein said illuminated reaction is a base extension reaction.

4. The method of claim 1, wherein the enzyme is a polymerase or a ligase.

5. The method of claim 1, wherein the reaction mixture further comprises a template nucleic acid molecule.

6. The method of claim 1, wherein the fluorescent or fluorogenic substrate comprises a nucleoside polyphosphate or analog thereof.

7. The method of claim 1, wherein at least one component of the reaction mixture is confined within a zero mode waveguide.

8. The method of claim 1, wherein illuminating the reaction mixture comprises illuminating the reaction for a period of time that is less than a photo-induced damage threshold period, wherein the photo-induced damage threshold period is lengthened in the presence of the photoprotective agent.

9. The method of claim 1, wherein the carboxylate salt is a potassium salt.

10. A method for increasing the accuracy of an illuminated sequencing reaction, the method comprising:

providing a reaction mixture comprising a polymerase, a template nucleic acid, and a fluorescent or fluorogenic nucleotide or nucleotide analog;

adding a photoprotective agent to the reaction mixture, wherein the photoprotective agent comprises a compound of a formula selected from the group consisting of;

11. The method of claim 10, further comprising the step of monitoring a reaction between the polymerase and the fluorescent or fluorogenic nucleotide or nucleotide analog while illuminating the reaction mixture.

12. The method of claim 10, wherein the photoprotective agent reduces an amount of blinking or photobleaching of a dye within the fluorescent or fluorogenic nucleotide or nucleotide analog resulting from excitation of the dye by the excitation illumination to an amount that is less than that which would occur absent the photoprotective agent.

13. The method of claim 10, wherein said illuminated sequencing reaction is a base extension reaction.

14. The method of claim 10, wherein at least one component of the reaction mixture is confined within a zero mode waveguide.

15. The method of claim 10, wherein the polymerase is confined within a zero mode waveguide.

16. The method of claim 10, wherein the carboxylate salt is a potassium salt.