Process for detection of DNA modifications and protein binding by a single molecule manipulation
First Claim
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1. A method for detecting a modified base within a double-stranded nucleic acid molecule, said method comprising the steps of:
- a) providing a nucleic acid hairpin molecule consisting of a double-stranded stem and a single-stranded loop, wherein the 5′ and
3′
ends of the nucleic acid hairpin molecule are bound to different surfaces;
b) applying a force to move one of the surfaces away from the other surface, thereby yielding a completely denatured hairpin molecule;
c) contacting the completely denatured hairpin molecule with an antibody that specifically binds to the modified base;
d) reducing the force to allow renaturation of the hairpin molecule in the presence of the antibody,wherein the antibody binding to the modified base causes a transient blockage of the renaturation of the hairpin molecule;
e) detecting the transient blockage of the renaturation of the hairpin molecule due to binding of the antibody to the modified base in the hairpin molecule; and
f) determining the position of the modified base within the double-stranded nucleic acid molecule by determining the position of the transient blockage.
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Abstract
The present invention relates to a method for determining whether a protein binds to a specific DNA sequence. This method is useful in particular for identifying modifications to the DNA sequence (e.g. methylations) via the binding of proteins that specifically recognize those modifications (e.g. antibodies), but also to identify the binding sequence on DNA of a variety of proteins.
16 Citations
15 Claims
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1. A method for detecting a modified base within a double-stranded nucleic acid molecule, said method comprising the steps of:
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a) providing a nucleic acid hairpin molecule consisting of a double-stranded stem and a single-stranded loop, wherein the 5′ and
3′
ends of the nucleic acid hairpin molecule are bound to different surfaces;b) applying a force to move one of the surfaces away from the other surface, thereby yielding a completely denatured hairpin molecule; c) contacting the completely denatured hairpin molecule with an antibody that specifically binds to the modified base; d) reducing the force to allow renaturation of the hairpin molecule in the presence of the antibody, wherein the antibody binding to the modified base causes a transient blockage of the renaturation of the hairpin molecule; e) detecting the transient blockage of the renaturation of the hairpin molecule due to binding of the antibody to the modified base in the hairpin molecule; and f) determining the position of the modified base within the double-stranded nucleic acid molecule by determining the position of the transient blockage. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for detecting a modified base within a double-stranded nucleic acid molecule, said method comprising the steps of:
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a) providing a nucleic acid hairpin molecule consisting of a double-stranded stem and a single-stranded loop, wherein the 5′ and
3′
ends of the nucleic acid hairpin molecule are bound to different surfaces;b) applying a force to move one of the surfaces away from the other surface, thereby yielding a completely denatured hairpin molecule; c) contacting the completely denatured hairpin molecule with a protein that specifically binds to the modified base, wherein the modified base is mismatched with the base on the other strand of the hairpin molecule; d) reducing the force to allow renaturation of the hairpin molecule in the presence of the protein; e) detecting blockage of the renaturation of the hairpin molecule due to binding of the protein to the modified base in the hairpin molecule; and f) determining the position of the blockage.
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15. A method for detecting a modified base within a double-stranded nucleic acid molecule, said method comprising the steps of:
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a) providing a nucleic acid hairpin molecule consisting of a double-stranded stem and a single-stranded loop, wherein the 5′ and
3′
ends of the nucleic acid hairpin molecule are bound to different surfaces;b) applying a force to move one of the surfaces away from the other surface, thereby yielding a completely denatured hairpin molecule; c) contacting the completely denatured hairpin molecule with a protein that specifically binds to the modified base, d) reducing the force to allow renaturation of the hairpin molecule in the presence of the protein; e) detecting blockage of the renaturation of the hairpin molecule due to binding of the protein to the modified base in the hairpin molecule; and f) determining the position of the blockage; wherein the protein is selected from the group consisting of a MutS dimer, Msh2/Msh6, and Msh2/Msh3, and wherein the method further comprises hybridizing a single-stranded nucleic acid that has a mismatch with the completely denatured hairpin molecule prior to step c).
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Specification