Rapid DNA Sequencing by Peroxidative Reaction

• US 20110020806A1
• Filed: 03/30/2010
• Published: 01/27/2011
• Est. Priority Date: 03/31/2009
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Karhanek, Miloslav, Pourmand, Nader, Munishkina, Larissa
• Application Number:
  US12/750,104
• Publication Number:
  US 20110020806A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Disclosed is a method of polynucleic acid (e.g., DNA) sequencing which is based on the generation of pyrophosphate (PPi) that occurs when a complementary base is incorporated into a growing DNA strand being synthesized on a template. The method utilizes a cascade of enzymatic reactions catalyzed by hypoxanthine-phosphoribosyl transferase, xanthine oxidase, and peroxidase in addition to DNA polymerase and apyrase. The last chemical step in the cascade of reactions is the oxidation of a material such as an electrode or luminol by hydrogen peroxide. This generates a detectable electrical or optical signal. This method is independent of luciferase, does not require dATP analogue, and is intended to improve precision and sensitivity of DNA sequencing, and to lessen the unsynchronized polymerization.

Claims

1. A method of detecting incorporation of a nucleotide into a polynucleotide, whereby PPi is generated, comprising the steps of:

(a) combining said PPi with a phosphotransferase to form an oxidatable compound;

(b) in a second step, oxidizing the oxidatable compound to form hydrogen peroxide;

(c) then contacting the hydrogen peroxide with a light emitting compound; and

(d) detecting a signal, caused by reaction of the hydrogen peroxide with the light emitting compound, as indicative of incorporation of the nucleotide.

2. The method of claim 1, using enzymes hypoxanthine-phosphoribosyl transferase in step a, xanthine oxidase in step b, and peroxidase in step c.

3. The method of claim 1 where the polynucleotide is DNA.

4. The method of claim 1 where the enzymes hypoxanthine-phosphoribosyl transferase in step a, xanthine oxidase in step b, and peroxidase in step c are added sequentially.

5. The method of claim 4 where the enzymes are simultaneously present in the reaction mixture when a PPi is generated.

6. The method of claim 1 where the light emitting compound is a luminol compound.

7. The method of claim 1 where the oxidatable compound is hypoxanthine.

8. The method of claim 7 where the oxidatable compound is oxidized with the use of xanthine oxidase.

9. The method of claim 8 where the contacting of hydrogen peroxide is accompanied by reacting with peroxidase.

10. The method of claim 9 where the oxidatable compound is luminol.

11. The method of claim 10 where the phosphotransferase is HPRT.

12. The method of claims 11 further comprising the step of adding a uricase enzyme for further converting a urate product derived from step (b) to produce hydrogen peroxide.

13. A method of detecting the incorporation of a nucleotide into a polynucleotide, whereby PPi is generated, comprising the steps of:

(a) combining said PPi with a phosphotransferase to form an oxidatable compound;

(b) in a second step, oxidizing the oxidatable compound to form hydrogen peroxide;

(c) then decomposing the hydrogen peroxide to form oxygen and protons; and

(d) detecting a signal in an electrode generated as a result of the generation of said protons, as indicative of incorporation of the nucleotide.

14. The method of claim 13 wherein decomposing of the hydrogen peroxide comprises oxidation of metal in an electrode.

15. The method of claim 13 wherein decomposing of the hydrogen peroxide generates a charge differential sensed by an electrode held at a set voltage.

16. A kit for sequencing DNA, comprising HPRT.

17. The kit of claim 16 further comprising xanthine oxidase.

18. The kit of claim 16 further comprising peroxidase.

19. The kit of claims 16 further comprising apyrase.

20. The kit of claims 16 further comprising DNA polymerase.

21. The kit of claims 16 further comprising RNA polymerase.

22. The kit of claims 16 further comprising uricase.