Assay methods using nicking endonucleases

A method for preparing and analyzing 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 flap region to form at least one double-stranded flap, to thereby prepare the target analyte,
• e. translocating the target analyte through a fluidic channel, the fluidic channel comprising at least a pair of detector electrodes defining a detection volume therein, and
• e. monitoring changes in an electrical property across a fluidic channel as the target analyte is translocated therethrough, the changes in the electrical property big indicative of double-stranded regions of the target analyte and of the single-stranded flap regions.

A method for preparing and analyzing 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;
• e. translocating the target analyte through a nanopore while two electrodes produce a measurable current parallel to movement of the target analyte;
• and
• f. monitoring changes in an electrical property across the 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 flap regions.

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, and
• d. coating the single-stranded flap with a binding moiety that selectively binds with single-stranded DNA to thereby prepare the target analyte;
• e. translocating the target analyte through a fluidic channel, the fluidic channel comprising at least a pair of detector electrodes defining a detection volume therein;
• and
• f. monitoring changes in an electrical property across the 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 and analyzing 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,
• g. translocating the target analyte through a fluidic channel, the fluidic channel comprising at least a pair of detector electrodes defining a detection volume therein, and
• h. monitoring changes in an electrical property across the 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 and analyzing 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,
• g. translocating the target analyte through a nanopore, while two electrodes produce a measurable current parallel to movement of the target analyte;
• and
• h. monitoring changes in an electrical property across the 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.