Methods and kits for methylation detection

• US 20060234252A1
• Filed: 07/01/2005
• Published: 10/19/2006
• Est. Priority Date: 07/02/2004
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Andersen, Mark R.
• Application Number:
  US11/173,569
• Publication Number:
  US 20060234252A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)

Abstract

Methods for determining the methylation state of at least one target nucleotide that employ a reaction catalyzed by a structure-specific nuclease, typically coupled with a ligation reaction are disclosed. By detecting the cleaved flap, a ligation product, a ligation product surrogate, a hybridization complex, or combinations thereof, one can infer the degree to which the corresponding target nucleotide is methylated. Certain of the disclosed methods are particularly useful for evaluating bisulfite-treated target sequences and determining the degree of target nucleotide methylation. The disclosed methods are well suited for rapidly analyzing a large number of target sequences, typically in one or more multiplex reactions. Kits for performing coupled nuclease and ligase methylation detection assays are also disclosed.

Claims

1. A method for determining the degree of methylation of a target nucleotide comprising,

(a) reacting (1) a target sequence with (2) a first cleavage probe set comprising (i) a first cleavage probe comprising a sequence that is complementary to a first target region and (ii) a second cleavage probe comprising a sequence that is complementary to a second target region and that is downstream from a flap portion, wherein the second target region is located 5′

of the first target region and overlaps the first target region by at least one nucleotide, under effective conditions for the first and second cleavage probes of the first cleavage probe set to anneal to the corresponding first and second target regions, respectively, forming a first hybridization complex;

(b) cleaving the flap portion of the second cleavage probe in the first hybridization complex to generate a cleaved flap and form a second hybridization complex comprising (1) the target sequence, (2) the first cleavage probe, and (3) an annealed fragment of the second cleavage probe having a 5′

-terminal nucleotide located adjacent to the 3′

-end of the annealed first cleavage probe;

(c) ligating the first cleavage probe to the annealed fragment of the second cleavage probe to generate a first ligation product and form a third hybridization complex comprising the target sequence and the first ligation product; and

(d) determining the degree of methylation of the target nucleotide.

2. The method of claim 1, further comprising:

(e) denaturing the third hybridization complex; and

(f) performing one or more additional cycles of steps (a) through (c) and optionally, step (e).

3. The method of claim 2, further comprising:

(a) combining (1) the first ligation product with (2) a second cleavage probe set comprising (i) a first cleavage probe comprising a sequence that is complementary to a first region of the ligation product and (ii) a second cleavage probe comprising a sequence that is complementary to a second region of the ligation product and that is downstream from a flap portion, wherein the second region of the ligation product is located 5′

of the first region on the ligation product and overlaps the first region of the ligation product by at least one nucleotide, under effective conditions for the first and second cleavage probes of the second cleavage probe set to anneal to the corresponding first and second regions of the ligation product, respectively, and form a fourth hybridization complex;

(b) subjecting the fourth hybridization complex to a cycle of (1) cleaving the flap portion of the second cleavage probe in the fourth hybridization complex to generate a cleaved flap and form a fifth hybridization complex comprising (i) the first ligation product, (ii) the first cleavage probe, and (iii) an annealed fragment of the second cleavage probe having a 5′

-terminal nucleotide located adjacent to the 3′

-end of the annealed first cleavage probe and (2) ligating the first cleavage probe to the annealed fragment of the second cleavage probe to generate a second ligation product and form a sixth hybridization complex comprising the first ligation product and the second ligation product; and

optionally,

(c) denaturing the sixth hybridization complex; and

(d) performing one or more additional cycles of steps (a) and (b), and optionally step (c).

4. The method of claim 2, further comprising:

(a) combining (1) the first ligation product with (2) a first ligation probe set comprising (i) a first ligation probe comprising an upstream first ligation product-binding portion and (ii) a second ligation probe comprising a downstream first ligation product-binding portion, under effective conditions for the first and second ligation probes to anneal to the first ligation product, to forming a seventh hybridization complex comprising the first ligation probe and the second ligation probe of the first ligation probe set and the first ligation product;

(b) ligating the first ligation probe to the second ligation probe to generate a third ligation product and form an eighth hybridization complex comprising the first ligation product and the third ligation product; and

(c) denaturing the eighth hybridization complex.

5. The method of claim 3, further comprising:

(a) combining (1) the second ligation product with (2) a second ligation probe set comprising (i) a first ligation probe comprising an upstream first ligation product-binding portion and (ii) a second ligation probe comprising a downstream first ligation product-binding portion, under effective conditions for the first and second ligation probes of the second ligation probe set to anneal to the second ligation product, forming a ninth hybridization complex comprising the first ligation probe and the second ligation probe of the second ligation probe set and the second ligation product;

(b) ligating the first ligation probe to the second ligation probe to generate a fourth ligation product and form a tenth hybridization complex comprising the second ligation product and the fourth ligation product;

(c) denaturing the tenth hybridization complex; and

(d) performing one or more additional cycles of steps (a) and (b), and optionally step (c).

6. A method for determining the degree of methylation of a target nucleotide comprising,

(a) reacting (1) a target sequence with (2) a first cleavage probe set comprising (i) a first cleavage probe that can hybridize with the target sequence and (ii) a second cleavage probe that (a) can hybridize with the target sequence downstream of the first cleavage probe and (b) contains a flap portion, under effective conditions for the first and second cleavage probes of the first cleavage probe set to hybridize with the target sequence, forming a first hybridization complex;

(b) cleaving the flap portion of the second cleavage probe in the first hybridization complex to generate a cleaved flap and form a second hybridization complex comprising (1) the target sequence, (2) the first cleavage probe, and (3) an annealed fragment of the second cleavage probe having a 5′

-terminal nucleotide located adjacent to the 3′

-end of the annealed first cleavage probe;

(c) ligating the first cleavage probe to the annealed fragment of the second cleavage probe to generate a first ligation product and form a third hybridization complex comprising the target sequence and the first ligation product;

(d) denaturing the third hybridization complex;

(e) performing one or more additional cycles of steps (a) through (c) and optionally, step (d); and

(f) determining the degree of methylation of the target nucleotide.

7. The method of claim 6, wherein the target sequence is modified using sodium bisulfite.

8. The method of claim 6, wherein a probe of the cleavage probe set comprises a Modification or a degenerate base.

9. The method of claim 8, wherein the Modification comprises a substituted hydrocarbon, a ribonucleotide, an amide bond, a glycosidic bond, an LNA, a nucleotide analog, a universal base, a groove binder, or combinations thereof.

10. The method of claim 6, wherein the ligating comprises a ligase or enzymatically active mutants or variants thereof.

11. The method of claim 6, wherein the second target region overlaps the first target region by more than one nucleotide.

12. The method of claim 6, wherein a probe comprises a reporter group, a hybridization tag, a mobility modifier, an affinity tag, a reporter probe-binding portion, a minor groove binder, or combinations thereof.

13. The method of claim 12, further comprising a reporter probe.

14. The method of claim 13, wherein the determining comprises detecting the reporter group of a ligation product, a ligation product surrogate, a reporter probe or at least part of a reporter probe, or combinations thereof, and evaluating the ligation ratio or threshold cycle during or after a plurality of cycles.

15. The method of claim 6, wherein the determining comprises a mobility-dependent analytical technique, a mass spectrometer, a Substrate, a real-time instrument, or combinations thereof.

16. The method of claim 6, wherein the target sequence, a cleavage probe, a ligation probe, or combinations thereof, is bound to a Substrate.

17. The method of claim 16, wherein the Substrate comprises a hybridization tag complement, a reporter group, an affinity tag, an aptamer, an antibody, or combinations thereof.

20. A method for determining the degree of methylation of a target nucleotide, comprising:

a step for interrogating the target nucleotide;

a step for generating a cleaved flap;

a step for generating a ligation product; and

a step for determining the degree of methylation of the target nucleotide.

18. The method of claim 20, wherein a hybridization complex is formed on a Substrate.

19. The method of claim 18, wherein the target sequence, the cleavage probe, the ligation probe, the hybridization complex, or combinations thereof, are detected on the Substrate.