Methods and Kits for Detecting an Enzyme Capable of Modifying a Nucleic Acid

• US 20090098539A1
• Filed: 05/18/2006
• Published: 04/16/2009
• Est. Priority Date: 05/18/2005
• Status: Active Grant

• Current Assignee: Iseao Technologies Limited
• Original Assignee: Iseao Technologies Limited
• Inventors: Stanley, John Christopher, Wilson, Stuart, Banin, Sharon
• Granted Patent: US 8,080,375 B2
• Time in Patent Office: Days
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/57434 of prostate

Abstract

An improved method of detecting an enzyme in a sample, which enzyme is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule, comprises the steps of allowing the sample to be tested for the presence of the enzyme to interact with the nucleic acid molecule; and testing for interaction of the enzyme with the nucleic acid molecule by detecting the novel nucleic acid molecule generated only in the presence of the enzyme. The preferred enzyme is a phosphatase. The methods have a number of applications, for example in enhancing the sensitivity of immunoassays, for detecting pathogen associated phosphatase, for diagnosing certain conditions and for detecting specific contaminants in a sample.

Claims

1. A method of detecting an enzyme in a sample, which enzyme is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule, the method comprising:

allowing the sample to be tested for the presence of the enzyme to interact with the nucleic acid molecule; and

testing for interaction of the enzyme with the nucleic acid molecule by detecting the novel nucleic acid molecule generated only in the presence of the enzyme.

2. The method according to claim 1 wherein the enzyme is a phosphatase capable of removing terminal phosphates from a nucleic acid molecule.

3. The method according to claim 2 wherein the terminal phosphates that are removed are present at the 3′

end of the nucleic acid molecule.

4. The method according to claim 1 wherein the nucleic acid molecule is a synthesised oligonucleotide which is 3′

end labelled with a phosphate group.

5. The method according to claim 1 wherein the enzyme is alkaline phosphatase or prostatic acid phosphatase.

6. The method according to claim 1 wherein the novel nucleic acid molecule that is detected is generated by ligation of the 3′

end of the nucleic acid molecule to the 5′

end of a further nucleic acid molecule.

7. The method according to claim 6 wherein the detection step is carried out by incubating the sample with a ligase and the further nucleic acid molecule and testing for the presence of the novel nucleic acid molecule following incubation.

19. The method according to claim 1 wherein the nucleic acid molecule comprises a single stranded DNA molecule in which a terminal 3′

phosphate is present and an overlapping single stranded DNA molecule which is blocked at the 3′

end such that it cannot be extended.

20. The method according to claim 19 wherein the novel nucleic acid molecule that is detected is generated by polymerisation using the nucleic acid molecule originally phosphorylated at the 3′

end to prime nucleic acid synthesis, provided the phosphatase is present in the sample to remove the 3′

terminal phosphate group, using the overlapping single stranded DNA as a template for synthesis.

21. The method according to claim 1 wherein the nucleic acid molecule comprises partially dsDNA in which terminal 3′

phosphates are present.

22. The method according to claim 21 wherein the dsDNA comprises a nick in each strand.

23. The method according to claim 21 wherein the novel nucleic acid molecule that is detected is generated by polymerisation using the nucleic acid molecule originally phosphorylated at the 3′

end to prime nucleic acid synthesis, provided the phosphatase is present in the sample to remove the 3′

terminal phosphate group, using the single stranded element as a template for synthesis.

48. The method according to claim 1 wherein the novel nucleic acid molecule is detected using nucleic acid amplification techniques.

114. The method according to claim 1 which is used for detection of a phosphatase associated with an infectious agent and comprises specific capture of the phosphatase and whereby detection of the novel nucleic acid molecule indicates the presence of the infectious agent.

115. The method according to claim 114 wherein the specific capture utilises a specific antibody.

116. The method according to claim 115 wherein the specific antibody is immobilized on a solid support.

117. The method according to claim 114 wherein the infectious agent is Aspergillus or Staphylococcus species.

8-18. -18. (canceled)

24-47. -47. (canceled)

49-53. -53. (canceled)

54. A method of detecting a contaminant in a sample comprising:

a) applying the sample to a solid support upon which is immobilised a reagent capable of binding to the contaminant in the presence of the same contaminant linked to an enzyme which is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule;

b) incubating under conditions which permit enzyme activity; and

c) determining the presence or level of the novel nucleic acid molecule, wherein an absence or low level of the novel nucleic acid molecule is indicative of a higher level of contaminant in the sample.

55. A method of detecting a contaminant in a sample comprising:

a) capture and separation of the contaminant via a specific binding reaction involving a reagent capable of specifically binding to the contaminant, wherein the reagent is linked to an enzyme which is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule;

b) adding to the separated contaminant the nucleic acid molecule;

c) incubating under conditions which permit enzyme activity; and

d) testing for interaction of the enzyme with the nucleic acid molecule by detecting the novel nucleic acid molecule generated only in the presence of the enzyme, wherein detection of the novel nucleic acid molecule indicates the presence of the contaminant in the sample.

56-71. -71. (canceled)

72. A method of diagnosing prostate cancer in a mammalian subject comprising

allowing a sample obtained from the subject under test to be tested for the presence of prostatic acid phosphatase (PAP) to interact with a nucleic acid molecule;

and

testing for interaction of PAP with the nucleic acid molecule by detecting a novel nucleic acid molecule produced by extension of the original nucleic acid molecule, whereby the presence of the novel nucleic acid molecule is indicative of the presence of prostatic acid phosphatase (PAP) in the sample and is taken as an indication that the subject has prostate cancer.

73. A method of diagnosing a disease associated with elevated serum alkaline phosphatase levels in a mammalian subject comprising

allowing a sample obtained from the subject to be tested for the presence of serum alkaline phosphatase to interact with a nucleic acid molecule;

and

testing for interaction of serum alkaline phosphatase with the nucleic acid molecule by detecting a novel nucleic acid molecule produced by extension of the original nucleic acid molecule, whereby the presence of the novel nucleic acid molecule is indicative of the presence of serum alkaline phosphatase in the sample and is taken as an indication that the subject has a disease associated with elevated serum alkaline phosphatase levels.

74-77. -77. (canceled)

78. A kit for detecting an enzyme capable of adding or removing a chemical moiety to or from a nucleic acid molecule, which thereby confers the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule, comprising:

a nucleic acid molecule which is capable of being acted upon by the enzyme; and

means for detecting the novel nucleic acid molecule produced only if the enzyme is present,

wherein the nucleic acid molecule;

(a) has a 3′

terminal phosphate group and additionally comprises a further nucleic acid molecule having a terminal 5′

phosphate group or

(b) is ds and has a 5′

overhang to which is attached a 5′

terminal phosphate group and additionally comprises a further ds nucleic acid molecule having a 5′

overhang and a terminal 5′

phosphate group attached to this, or

(c) comprises a single stranded DNA molecule in which a terminal 3′

phosphate is present and an overlapping single stranded DNA molecule which is blocked at the 3′

end such that it cannot be extended, or

(d) comprises dsDNA in which terminal 3′

phosphates are present and which has a single stranded element to allow priming of nucleic acid synthesis if the terminal phosphates are removed by the phosphatase in the sample, or

(e) comprises dsDNA with one strand having a 5′

overhang and wherein the shorter strand has a 3′

terminal phosphate group attached.

102. The kit according to claim 78 further comprising an antibody selective for an infectious agent-specific phosphatase.

79-101. -101. (canceled)

103. (canceled)

104. A kit for detection of a contaminant in a sample comprising:

(i) a reagent capable of specifically binding to the contaminant, wherein the reagent is linked to an enzyme which is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule; and

(ii) a nucleic acid molecule which is capable of being acted upon by the enzyme.

105. A kit for detecting a contaminant in a sample comprising:

(i) a conjugate of the same contaminant as that which is to be detected and an enzyme which is capable of adding or removing a chemical moiety to or from a nucleic acid molecule, thereby conferring the nucleic acid molecule with the ability to be extended to generate a novel detectable nucleic acid molecule; and

(ii) a nucleic acid molecule which is capable of being acted upon by the enzyme.

106-113. -113. (canceled)