Binary signaling assay using a split-polymerase

• US 7,659,069 B2
• Filed: 08/31/2007
• Issued: 02/09/2010
• Est. Priority Date: 08/31/2007
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Belyaev, Alexander, Monell, Craig Robert, Sorge, Joseph A.
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US11/897,892
• Publication Number:
  US 20090061426A1
• Time in Patent Office: 893 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The present invention provides methods, kits and compositions for the detection of an analyte. In the methods of the invention, a complex is formed between two or more analyte specific probes (ASP) and an analyte. The analyte specific probes each have a portion of a polymerase which interact to form a functional polymerase complex upon binding of the ASP to the analyte. The functional polymerase complex then generates a detectable signal which is indicative of the presence and/or amount of the analyte in the sample.

Claims

1. A method for detecting an analyte in a sample, said method comprising:

a. forming a reaction mixture comprising;

a first analyte specific probe and a second analyte specific probe, wherein said first analyte specific probe comprises a first binding moiety and a first portion of a first polymerase and wherein said second analyte specific probe comprises a second binding moiety and a second portion of said first polymerase,

a modified polynucleotide template,

a first primer complementary to said polynucleotide template,

a second polymerase, and

a second primer complementary to an amplification template;

b. incubating said reaction mixture so as to permit;

said first and said second binding moieties to bind to said analyte such that said first and said second portions of said first polymerase interact to form a polymerase complex,

annealing of said first primer to said modified polynucleotide template, and

extending said primer by said polymerase complex thereby synthesizing said amplification template;

c. annealing said second primer to said amplification template and extending said primer with said second polymerase and amplifying said amplification template with said second polymerase, wherein said second polymerase is unable to amplify the modified polynucleotide template under the conditions of the first primer extension reaction; and

d. detecting said amplified amplification template wherein detection of said amplified amplification template is indicative of the presence or amount of said analyte in a sample.

12. The method of claim 1, wherein the first and second binding moieties are antibodies.

13. The method of claim 1, wherein the polymerase complex is Klenow or T4.

14. The method of claim 12, wherein the antibodies are coupled to the first and second portions of the first polymerase by a streptavidin-biotin interaction.

15. The method of claim 14, wherein the antibodies are biotinylated and the first and second portions of the first polymerase are coupled to streptavidin.

16. The method of claim 1, wherein the modified polynucleotide template comprises one or more deoxyuracils and/or one or more 2′

O-methyl modifications.

2. A method for detecting an analyte in a sample, said method comprising:

a. contacting a sample with a reaction mixture so as to permit;

binding of a first analyte specific probe and a second analyte specific probe to an analyte so as to form a polymerase complex, wherein said first analyte specific probe comprises a first binding moiety and a first portion of a first polymerase and wherein said second analyte specific probe comprises a second binding moiety and a second portion of said first polymerase,

annealing a first primer to a modified polynucleotide template, and

extending said primer with said polymerase complex thereby synthesizing an amplification template;

b. annealing a second primer to said amplification template and extending said second primer with a second polymerase and amplifying said amplification template with said second polymerase, wherein said second polymerase is unable to amplify the modified polynucleotide template under the conditions of the first primer extension reaction; and

c. detecting said amplified amplification template, wherein detection of said amplified amplification template is indicative of the presence or amount of said analyte in a sample.

3. The method of claim 2, wherein said binding moiety is selected from the group consisting of a monoclonal antibody, polyclonal antibody, lectin, cell surface receptor, receptor ligand, peptide, carbohydrate, aptamer, biotin, streptavidin, avidin, protein A and protein G.

4. The method of claim 2, wherein said analyte is selected from the group consisting of a protein, oligonucleotide, cell surface receptor and receptor ligand.

5. The method of claim 2, wherein said detecting is performed during a real-time PCR reaction.

6. The method of claim 2, wherein said detecting comprises a nucleic acid cleavage reaction.

7. The method of claim 2, wherein said detecting further comprises a labeled probe that anneals downstream of said second primer.

8. The method of claim 2, wherein said first portion of said first polymerase comprises the amino acid sequence of SEQ ID NO:

2 and said second portion of said first polymerase comprises the amino acid sequence of SEQ ID NO;

3.

9. The method of claim 2, wherein said detecting comprises detecting an amplification product of the extended second primer.

10. The method of claim 2, wherein said second polymerase is Pfu DNA polymerase or hot start Taq DNA polymerase.

11. The method of claim 2, wherein said reaction mixture is treated with uracil deglycosylase prior to extending said second primer with said second polymerase.

17. The method of claim 2, wherein the first and second binding moieties are antibodies.

18. The method of claim 2, wherein the polymerase complex is Klenow or T4.

19. The method of claim 17, wherein the antibodies are coupled to the first and second portions of the first polymerase by a streptavidin-biotin interaction.

20. The method of claim 19, wherein the antibodies are biotinylated and the first and second portions of the first polymerase are coupled to streptavidin.

21. The method of claim 2, wherein the modified polynucleotide template comprises one or more deoxyuracils and/or one or more 2′

O-methyl modifications.