Methods, systems and compositions for monitoring enzyme activity and applications thereof

• US 7,563,574 B2
• Filed: 03/31/2006
• Issued: 07/21/2009
• Est. Priority Date: 03/31/2006
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US11/394,637
• Publication Number:
  US 20070231804A1
• Time in Patent Office: 1,208 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Methods of monitoring enzyme mediated reactions, and particularly nucleic acid synthesis reactions such as pyrosequencing methods that employ enzymatic reporter systems. The methods and systems provide elevated signal levels as compared to conventional pyrosequencing processes, and/or mediate de-phasing of sequencing analyses employing pyrosequencing or other “sequencing by synthesis” methods.

Claims

1. A method of monitoring an activity of an enzyme that is capable of producing pyrophosphate from a nucleoside triphosphate, comprising:

contacting the enzyme with an unlabeled nucleoside polyphosphate having more than three phosphate groups; and

detecting polyphosphate released from the nucleoside polyphosphate as an indicator of enzyme activity, wherein the step of detecting the polyphosphate comprises;

contacting the polyphosphate with an enzyme system capable of producing two or more ATP molecules from each polyphosphate molecule released; and

detecting the ATP produced.

2. The method of claim 1, wherein the enzyme comprises a nucleic acid polymerase.

3. The method of claim 1, wherein the enzyme system in the detecting step comprises an ATP:

Polyphosphate phosphotransferase enzyme, ADP and a sulfurylase enzyme.

4. The method of claim 1, wherein the enzyme system in the detecting step comprises a luciferase enzyme.

5. The method of claim 1, wherein the step of detecting ATP comprises contacting the ATP with a reporter system that is responsive to ATP.

6. The method of claim 5, wherein the reporter system comprises a luciferase enzyme reporter system.

7. The method of claim 1, wherein the nucleoside polyphosphate is selected from a nucleoside tetraphosphate, a nucleoside pentaphosphate, a nucleoside hexaphosphate or an analog thereof.

8. A method of monitoring an activity of a polymerase enzyme, comprising:

contacting the polymerase enzyme with a nucleoside polyphosphate comprising more than three phosphate groups, wherein the polymerase releases a polyphosphate group having at least three phosphates;

converting the polyphosphate group into at least two ATP molecules for each polyphosphate group released; and

detecting an amount of ATP produced in the converting step as an indication of an activity of the polymerase enzyme.

9. A method of monitoring an activity of an enzyme system that is capable of releasing pyrophosphate from a nucleoside triphosphate, comprising:

contacting the enzyme system with a nucleoside polyphosphate having more than three phosphate groups, and

contacting polyphosphate released from the nucleoside polyphosphate with an enzyme system capable of producing two or more ATP molecules from each polyphosphate molecule; and

detecting ATP produced.