Methods of detecting sequence-specific DNA binding proteins

• US 20090208927A1
• Filed: 10/16/2006
• Published: 08/20/2009
• Est. Priority Date: 03/28/2002
• Status: Active Grant

• Current Assignee: Marligen Biosciences, Inc. (Origene Technologies Incorporated)
• Original Assignee: Marligen Biosciences, Inc. (Origene Technologies Incorporated)
• Inventors: Lazar, James G., Carlson, David P., Clausen, Peter A.
• Granted Patent: US 7,771,940 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: G01N 2333/4703 Regulators; Modulating activity
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

Compositions and methods are provided for detecting and measuring DNA-binding proteins. The compositions and methods permit the simultaneous or near-simultaneous detection of multiple DNA-binding proteins in a multiplex or array format, and can be used to generate profiles of DNA binding activity by proteins, specifically, transcription factors. Multiple protein-DNA binding events in a single sample may be detected and quantitated in a high-throughput, format.

Claims

1. A method for detecting a plurality of sequence specific DNA binding proteins, comprising:

(a) contacting a plurality of detection duplexes, wherein each of said, detection duplexes comprises (i) a first oligonucleotide comprising a distinguishable capture tag, wherein said capture tag is a nucleic acid sequence of natural nucleotides linked by natural linkages between nucleotides, and (ii) a second oligonucleotide that is complementary to said first oligonucleotide, wherein said second oligonucleotide comprises a detectable label, with a cellular or tissue extract suspected of containing a plurality of sequence specific DNA binding proteins for a time sufficient to permit, sequence-specific binding between said duplex and said binding proteins;

(b) contacting the mixture from step (a) with a cleavage reagent that is capable of cleaving said detection duplex, wherein cleavage of said detection duplex is inhibited by binding of said DNA binding protein to said duplex;

(c) capturing the resulting mixture on a solid support by hybridizing said capture tag to a complementary oligonucleotide on said solid support and

(c) detecting the inhibition of said cleavage by said DNA binding protein by detecting fee presence or absence of the label on said solid support.

30. The method according to claim 1, wherein said cleavage reagent is a sequence specific cleavage reagent.

34. The method according to claim 1 wherein said nuclease is an exonuclease.

2-20. -20. (canceled)

21. The method according to claim wherein said label comprises biotin, digoxigenin, a peptide, a protein, a small molecule, a radioactive isotope, a fluorescent moiety, a chemiluminescent label, a bioluminescent label, or an enzyme capable of generating a signal.

22. The method according to claim 21, wherein each detection duplex is captured at a predetermined position on a solid surface.

23. The method according to claim 21 wherein each detection duplex is captured on a distinguishable bead.

24. The method according to claim 22, wherein said cleavage reagent, is a non-sequence specific cleavage reagent.

25. The method according to claim 24, wherein said cleavage reagent is an endonuclease.

26. The method according to claim 25, wherein said cleavage reagent has a preference for double-stranded nucleic acids.

27. The method according to claim 23, wherein said cleavage reagent is a non-sequence specific cleavage reagent.

28. The method according to claim 27, wherein said cleavage reagent is an endonuclease.

29. The method according to claim 28, wherein said cleavage reagent has a preference for double-stranded nucleic acids.

31. The method according to claim 21, wherein said cleavage reagent is a sequence specific cleavage reagent.

32. The method according to claim 22, wherein said cleavage reagent is a sequence specific cleavage reagent.

33. The method according to claim 23, wherein said cleavage reagent is a sequence specific cleavage reagent.

35. The method according to claim 21 wherein said nuclease is an exonuclease.

36. The method according to claim 22 wherein said nuclease is an exonuclease.

37. The method according to claim 23 wherein said nuclease is an exonuclease.