Assay methods using nicking endonucleases
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.
1. 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 being indicative of double-stranded regions of the target analyte and of the single-stranded flap regions.
2. The method of claim 1 , wherein the nicking endonuclease comprises one or more endonucleases selected from the group consisting of Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.
3. The method of claim 1 , wherein the first base extension reaction comprises contacting the first DNA strand with a polymerase, one or more nucleotides, a ligase, or any combination thereof.
4. The method of claim 1 , wherein the second base extension reaction comprises contacting at least one flap region with a polymerase, one or more nucleotides, a ligase, or any combination thereof.
5. The method of claim 1 , wherein the fluidic channel comprises a micro-channel or a nano-channel.
6. The method of claim 1 , further comprising:
differentiating between double-stranded and flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
7. The method of claim 1 , further comprising:
coating the target analyte with a binding moiety.
8. The target analyte of claim 7 , wherein the binding moiety comprises a protein.
9. The target analyte of claim 8 , wherein said protein includes one or more proteins selected from the group consisting of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.
10. The method of claim 1 , further comprising:
differentiating between double-stranded and flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
11. The method of claim 1 , wherein the electrical property is an electrical potential or a current.
12. 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.
13. The method of claim 12 , further comprising:
differentiating between double-stranded and flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
14. The method of claim 12 , wherein the electrical property is an electrical potential or a current.
15. 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.
16. The method of claim 15 , wherein the electrical property is an electrical potential or a current.
17. The method of claim 15 , wherein the nicking endonuclease comprises one or more endonucleases selected from the group consisting of Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.
18. The method of claim 15 , wherein the base extension reaction comprises contacting the first DNA strand with a polymerase, one or more nucleotides, a ligase, or any combination thereof.
19. The method of claim 15 , wherein the binding moiety comprises a protein.
20. The method of claim 15 , wherein the fluidic channel comprises a micro-channel or a nano-channel.
21. The method of claim 15 , further comprising:
differentiating between double-stranded and single-stranded flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
22. 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 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 single-stranded flap regions.
23. The method of claim 22 , further comprising:
differentiating between double-stranded and single-stranded flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
24. The method of claim 22 , wherein the electrical property is an electrical potential or a current.
25. 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.
26. The method of claim 25 , wherein the single-stranded extension added to the double-stranded flap is a single-stranded polyT extension.
27. The method of claim 26 , wherein the probes comprise polyA oligomers.
28. The method of claim 25 , wherein the single-stranded extension added to the double-stranded flap is a single-stranded polyA extension.
29. The method of claim 28 , wherein the probes comprise polyT oligomers.
30. The method of claim 25 , wherein the probes are tagged.
31. The method of claim 25 , wherein the single-stranded extension is added to the double-stranded flap using a terminal transferase.
32. The method of claim 25 , wherein the single-stranded extension is at least 100 bases in length.
33. The method of claim 25 , wherein the fluidic channel comprises a micro-channel or a nano-channel.
34. The method of claim 25 , further comprising:
differentiating between double-stranded and single-stranded flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
35. The method of claim 25 , wherein the electrical property is an electrical potential or a current.
36. 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.
37. The method of claim 36 , further comprising:
differentiating between double-stranded and single-stranded flap regions of the target analyte based, at least in part, on the detected changes in the electrical property, to thereby determine nick locations and map at least a portion of the double-stranded DNA template.
38. The method of claim 36 , wherein the electrical property is an electrical potential or a current.