Assay methods using nicking endonucleases
First Claim
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1. A method for preparing and analyzing a target analyte, the method comprising:
- a. providing a double-stranded DNA template having a first and a second DNA strand, each DNA strand having a 5′
end and a 3′
end,b. contacting the double-stranded DNA template with a nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand,c. conducting a base extension reaction on the first DNA strand along a corresponding region of the second DNA strand, said reaction starting at the nick and progressing toward the 3′
end of the first DNA strand to thereby form a single-stranded flap on the double-stranded DNA template adjacent to the sequence-specific nicking location, to thereby prepare the target analyte, andd. monitoring changes in an electrical property across a nanopore as the target analyte is translocated therethrough, the changes in the electrical property being indicative of double-stranded regions of the target analyte and of the single-stranded flap regions.
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Abstract
Assay methods and apparatus for the analysis of biopolymers are disclosed. The assays employ nicking endonucleases to enable the generation of flaps on target biomolecules which are detected in nanopore or fluidic channel devices. Identification of flap locations enables a map of the target biomolecule to be derived.
234 Citations
36 Claims
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1. A method for preparing and analyzing a target analyte, the method comprising:
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a. providing a double-stranded DNA template having a first and a second DNA strand, each DNA strand having a 5′
end and a 3′
end,b. contacting the double-stranded DNA template with a nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand, c. conducting a base extension reaction on the first DNA strand along a corresponding region of the second DNA strand, said reaction starting at the nick and progressing toward the 3′
end of the first DNA strand to thereby form a single-stranded flap on the double-stranded DNA template adjacent to the sequence-specific nicking location, to thereby prepare the target analyte, andd. monitoring changes in an electrical property across a nanopore as the target analyte is translocated therethrough, the changes in the electrical property being indicative of double-stranded regions of the target analyte and of the single-stranded flap regions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method for preparing and analyzing a target analyte, the method comprising:
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a. providing a double-stranded DNA template having a first and a second DNA strand, each DNA strand having a 5′
end and a 3′
end,b. contacting the double-stranded DNA template with a nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand, c. conducting a base extension reaction on the first DNA strand along a corresponding region of the second DNA strand, said reaction starting at the nick and progressing toward the 3′
end of the first DNA strand to thereby form a single-stranded flap on the double-stranded DNA template adjacent to the sequence-specific nicking location, to thereby prepare the target analyte, andd. monitoring changes in an electrical property across a fluidic channel as the target analyte is translocated therethrough, the changes in the electrical property being indicative of double-stranded regions of the target analyte and of the single-stranded flap regions. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method for preparing a target analyte, the method comprising:
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a. providing a double-stranded DNA template having first and second DNA strands, each strand having a 5′
end and a 3′
end,b. contacting the template with a nicking endonuclease to form nicks at sequence-specific locations on the first DNA strand, c. conducting a first base extension reaction on the first DNA strand along the corresponding region of the second DNA strand, said reaction starting at each nick and progressing toward the 3′
end of the first DNA strand to thereby form single-stranded flap regions on the double-stranded DNA template adjacent to the sequence-specific nicking locations,d. conducting a second base extension reaction on at least one single-stranded flap region to form at least one double-stranded flap, e. adding a single-stranded extension to the double-stranded flap, and f. hybridizing one or more probes to the single-stranded extension, to thereby prepare the target analyte, wherein the probes are tagged using gold particles.
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Specification