METHODS AND KITS FOR DETECTION OF METHYLATION STATUS

• US 20140363815A1
• Filed: 12/13/2012
• Published: 12/11/2014
• Est. Priority Date: 12/13/2011
• Status: Active Grant

• Current Assignee: Oslo Universitetssykehus Hf
• Original Assignee: Oslo Universitetssykehus Hf
• Inventors: Dahl, John Arne, Robertson, Adam Brian, Klungland, Arne, Ellevog, Linda
• Granted Patent: US 10,023,909 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC) and/or 5-methylcytosine (5meC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC and/or 5meC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA

Claims

1. A process for detecting 5-methylated and/or other modified cytosine residues in a nucleic acid sample comprising:

a) replicating said nucleic acid sample under conditions such that 5-methylated cytosine residues are maintained and said other modified cytosine residues are diluted;

b) treating said replicated nucleic acid sample to convert unmodified cytosine residues to a uracil or thymidine residues; and

c) reading the sequence of said replicated nucleic acid sample wherein 5-hydroxymethylated cytosine residues are identified as residues that are read by sequencing as a thymidine or uracil residue in said replicated nucleic acid sample.

2. The process of claim 1, wherein said nucleic acid sample is divided into at least first and second portions and said replicating and treating steps are performed on said first portion, and comparing the sequence of said first nucleic acid portion with the sequence of said second nucleic acid portion, wherein said other modified cytosine residues are identified as residues that are read by sequencing as a uracil or thymidine residue in said first nucleic acid portion and as a cytosine residue at the corresponding position in said second nucleic acid portion and wherein 5-methylated cytosine residues are identified as residues that are read as cytosine residues in both of said first and second nucleic acid portions.

3. The process of claim 2, wherein said treating said first and second portions to convert unmodified cytosine residues to thymidine residues further comprises treating said first and second nucleic acid portions with bisulfite to convert unmodified cytosine residues to uracil resides and replicating said first and second nucleic acid portions with a polymerase to convert said uracil residues into thymidine residues.

4. The process of claim 2, wherein said replicating of said first portion further comprises:

a) replicating said nucleic acid with a tagged primer to provide tagged replicated nucleic acid;

b) treating said tagged replicated nucleic acid strands with a DNA methyltransferase to provide tagged 5-methylcytosine-modified replicated nucleic acid;

c) isolating said tagged 5-methylcytosine-modified replicated nucleic acid;

d) treating said isolated tagged 5-methylcytosine-modified replicated nucleic acid with bisulfite to convert unmodified cytosine residues to uracil residues; and

e) replicating said isolated tagged bisulfite-treated nucleic acid with a polymerase to provide a first bisulfite treated nucleic acid portion.

5. The process of claim 4, wherein said tagged primer is a biotinylated primer.

6. The process of claim 1, wherein said other modified cytosine residues are selected from the group consisting of 5-hydroxymethyl cytosine, b-glu-5-hydroxymethyl cytosine, 5-formyl-cytosine and 5-carboxycytosine.

7. The process of claim 1, wherein said replicating said first portion under conditions such that 5-methylated cytosine residues are maintained and 5-hydroxymethylated cytosine residues are diluted comprises replicating said nucleic acid with a polymerase to provide replicated nucleic acid and treating said replicated nucleic acid with an enzyme to 5-methylate cytosine residues.

8. The process of claim 1, wherein said steps of replication and treating with an enzyme are performed one or more times.

9. The process of claim 1, wherein said steps of replication and treating with an enzyme are repeated 5 or more times.

10. The process of claim 1, wherein said steps of replication and treating with an enzyme are repeated 7 or more times.

11. The process of claim 1, wherein said steps of replication and treating with an enzyme are repeated 10 or more times.

12. The process of claim 1, wherein said steps of replication and treating with an enzyme are performed from about 1 to about 20 times or more.

13. The process of claim 1, wherein said replication is by a polymerase chain reaction.

14. The process of claim 1, wherein said replication is by a primer extension reaction.

15. The process of claim 1, wherein said replication utilizes a biotinylated primer.

16. The process of claim 1, wherein said enzyme is a DNA methyltransferase.

17. The process of claim 16, wherein said DNA methyltransferase is selected from the group consisting of mouse DNMT1, human DNMT1 and M.SssI DNMT.

18. The process of claim 1 further comprising the step of modifying 5-hydroxymethylated cytosine residues in said samples to prevent methylation of said 5-hydroxymethylated cytosine residues.

19. The process of claim 18, wherein said 5-hydroxymethylated cytosine residues are modified by addition of a blocking group.

20. The process of claim 19, wherein said blocking group is selected from the group consisting of beta-glucose, alpha-glucose);

6-O-β

-D-glucopyranosyl-D-glucose, 6-O-alpha-D-glucopyranosyl-D-glucose;

keto-glucose;

azide-glucose; and

modified versions thereof.

21. The process of claim 1, wherein said nucleic acid sample is selected from the group consisting of human, plant, mouse, rabbit, hamster, primate, fish, bird, cow, sheep, pig, viral, bacterial and fungal nucleic acid samples.

22. The process of claim 1, further comprising comparing the presence of 5-hydroxymethylcytosine and/or 5-methylcytosine in said nucleic acid in said sample to a reference standard, wherein an increased or decreased level of 5-hydroxymethylcytosine and/or 5-methylcytosine in said nucleic acid is indicative of the presence of a disease or of the probable course of a disease.

23. The process of claim 1, further comprising the step of providing a diagnoses or prognoses based on an increased or decreased level of 5-hydroxymethylcytosine and/or 5-methylcytosine in said nucleic acid as compared to a reference standard.

24. The process of claim 23, wherein said disease is cancer.

25. The process of claim 1, wherein said nucleic acid sample is genomic DNA.

40. A process for predicting a predisposition to a disease in a subject, diagnosing a disease in a subject, predicting the likelihood of recurrence of disease in a subject, providing a prognosis for a subject with a disease, or selecting a subject with a disease for treatment with a particular therapy, comprising:

a) providing a genomic DNA sample from said subject; and

b) detecting the methylation status of predetermined portions of said genomic DNA sample by the process of claim 1,

wherein an altered level of 5-hydroxymethylcytosine and/or 5-methylcytosine methylation of said predetermined portions of said genomic DNA to a reference methylation status provides an indication selected from the group consisting of an indication of a predisposition of the subject to a disease, an indication that the subject has a disease, an indication of the likelihood of recurrence of a disease in the subject, an indication of survival of the subject, and an indication that the subject is a candidate for treatment with a particular therapy.

41. The process of claim 40, wherein said disease is a cancer.

42. The process of claim 40, wherein said subject is a human.

26. A process for detecting methylated and hydroxymethylated cytosine residues in a nucleic acid sample comprising:

a) dividing said sample into at least first and second untreated portions;

b) replicating said first portion with a tagged primer and a polymerase to provide parent and tagged replicated nucleic acid;

c) treating said parent and said tagged replicated nucleic acid strands with a DNA methyltransferase to provide tagged 5-methylcytosine-modified replicated nucleic acid;

d) isolating said tagged 5-methylcytosine-modified replicated nucleic acid;

e) treating said isolated tagged 5-methylcytosine-modified replicated nucleic acid with bisulfite to convert unmodified cytosine residues to uracil residues;

f) replicating said isolated tagged bisulfite-treated nucleic acid with a polymerase to provide a first bisulfite treated nucleic acid portion;

g) sequencing said first bisulfite treated nucleic acid portion;

h) treating said second portion with bisulfite to convert unmodified cytosine residues to uracil residues;

i) replicating said bisulfite-treated nucleic acid with a polymerase to provide a second bisulfite treated nucleic acid portion;

j) sequencing said second bisulfite treated nucleic acid portion; and

k) comparing the sequence of said first bisulfite treated nucleic acid portion with the sequence of said second bisulfite treated portion, wherein 5-hydroxymethylated cytosine residues are identified as residues that are read by sequencing as a uracil or thymidine residue in said first bisulfite treated nucleic acid portion and as a cytosine residue at the corresponding position in said second bisulfite treated nucleic acid portion and wherein 5-methylated cytosine residues are identified as residues that are read as cytosine residues in said first and second bisulfite treated portions.

27-39. -39. (canceled)