Assay Methods Using Nicking Endonucleases
AssigneeNABsys, Inc.
InventorJohn S. Oliver
Life of this patent
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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.
Claims80
A method for preparing 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, and
- d. coating the double-stranded DNA template with a binding moiety that enhances electrical detection of the double-stranded DNA template and the single-stranded flap, to thereby prepare the target analyte.
A target analyte comprising a double-stranded DNA template having at least one single-stranded, flap region extending therefrom, at least a portion of the double-stranded DNA template and the single-stranded flap region being coated with a binding moiety that enhances electrical detection of the double-stranded DNA template and the single-stranded flap region.
A method for preparing a target analyte, the method comprising:
- 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, and
- d. conducting a second base extension reaction on at least one flap region to form at least one double-stranded flap, to thereby prepare the target analyte.
A target analyte comprising a double-stranded DNA template having one or more double-stranded flap regions extending therefrom.
A method for preparing 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, and
- d. 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.
A method for preparing 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, and
- d. 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.
A method for preparing 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, and
- d. coating the single-stranded flap with a binding moiety that selectively binds with single-stranded DNA to enhance electrical detection of the single-stranded flap, to thereby prepare the target analyte.
A target analyte comprising a double-stranded DNA template having at least one single-stranded, flap region extending therefrom, the single-stranded flap region being coated with a binding moiety that enhances electrical detection thereof.
A method for preparing a target analyte, the method comprising:
- 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.
A target analyte comprising a double-stranded DNA template having one or more double-stranded flap regions extending therefrom, at least one double-stranded flap region having a single-stranded extension added thereto.
Dependencies are inferred from the claim text.