COMPOSITION AND METHODS RELATED TO MODIFICATION OF 5-METHYLCYTOSINE (5-mC)

• US 20140322707A1
• Filed: 04/06/2012
• Published: 10/30/2014
• Est. Priority Date: 04/06/2011
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: He, Chuan, Zhang, Liang, Lu, Xingyu
• Granted Patent: US 9,611,510 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The present invention relates to methods and compositions for detecting, evaluating, and/or mapping 5-methyl-modified and/or 5-hydroxylmethyl-modified cytosine bases within a nucleic acid molecule.

Claims

1. A method for detecting 5-methylcytosine in a nucleic acid comprising converting 5-methylcytosine to 5-hydroxymethylcytosine, 5-carboxycytosine, or 5-formylcytosine with a methylcytosine dioxygenase or the catalytic domain of a methylcytosine dioxygenase and sequencing the nucleic acid to detect 5-mC detecting 5-hydroxymethylcytosine.

3. The method of claim 1, wherein the methylcytosine dioxygenase is TET1, TET2, or TET3, or a homologue thereof.

4. The method of claim 1, wherein the methylcytosine dioxygenase comprises amino acids 1367-2039 of SEQ ID NO:

1.

5. The method of claim 1, further comprising modifying 5-hydroxymethylcytosine with a detectable label or a detectable functional group.

8. The method of claim 1, wherein the 5-hydroxymethylcytosine is converted from 5-methylcytosine and modified by a glucose or modified glucose molecule, comprising a process of incubating the nucleic acid molecule with a methylcytosine dioxygenase, a β

-glucosyltransferase and a glucose or modified glucose molecule.

18. The method of claim 1, further comprising modifying 5-hydroxymethylcytosine in the nucleic acid composition prior to converting 5-methylcytosine to 5-hydroxymethylcytosine.

19. The method of claim 18, wherein 5-hydroxymethylcytosine is modified with a glucose or a modified glucose.

20. The method of claim 18, further comprising detecting modified 5-hmC 19, wherein 5-hydroxymethylcytosine is modified by a process comprising incubating the nucleic acid molecule with a β

-glucosyltransferase and a glucose or a modified glucose.

23. The method of claim 18, further comprising modifying 5-hydroxymethylcytosine with a first detectable label or a first detectable functional group.

37. The method of claim 1, wherein the nucleic acid molecule is genomic DNA.

2. (canceled)

6-7. -7. (canceled)

9-17. -17. (canceled)

21-22. -22. (canceled)

24-36. -36. (canceled)

38-79. -79. (canceled)

80. A method for distinguishing 5-hydroxymethylcytosine (5-hmC) from 5-methylcytosine (5-mC) in a nucleic acid molecule comprising:

a) modifying 5-hydroxymethylcytosine in the nucleic acid molecule;

b) treating the nucleic acid molecule with methylcytosine dioxygenase to oxidize 5-mC; and

, under conditions to convert 5-methylcytosine to 5-carboxylcytosine;

c) then sequencing the treated nucleic acid to identify 5-hmC and 5-mC using bisulfite.

82. The method of claim 80, wherein the methylcytosine dioxygenase is TET1, TET2, or TET3, or a homologue thereof.

85. The method of claim 80, wherein 5-hydroxymethylcytosine is modified with a glucose or a modified glucose.

86. The method of claim 85, wherein the 5-hydroxymethylcytosine is modified by a process comprising incubating the nucleic acid molecule with β

-glucosyltransferase and a glucose or modified glucose molecule.

91. The method of claim 80, wherein the nucleic acid molecule is genomic DNA.

81. (canceled)

83-84. -84. (canceled)

87-90. -90. (canceled)

92-110. -110. (canceled)

111. A method for detecting 5-methylcytosine (5-mC) in a nucleic acid molecule comprising

converting 5-mC to a modified 5-mC using enzymatic modification by a methylcytosine dioxygenase;

and,

detecting modified 5-mC by sequencing.

112. The method of claim 111, wherein the methylcytosine dioxygenase is TET1, TET2, TET3 or a homologue thereof.

113. The method of claim 114, wherein the methylcytosine dioxygenase comprises amino acids 1367-2039 of SEQ ID NO:

1.

114. The method of claim 111, wherein the modified 5-mC comprises a detectable label or detectable functional group.

115. The method of claim 114, wherein the detectable label is fluorescent, radioactive, enzymatic, electrochemical, or colorimetric.

116. The method of claim 111, further comprising modifying 5-hydroxymethylcytosine in the nucleic acid composition prior to converting 5-methylcytosine to a modified 5-mC.

117. The method of claim 116, further comprising identifying 5-hmC in the nucleic acid by sequencing.

118. The method of claim 116, wherein 5-hydroxymethylcytosine is modified with a glucose or a modified glucose.

119. The method of claim 118, wherein 5-hydroxymethylcytosine is modified by a process comprising incubating the nucleic acid molecule with a β

-glucosyltransferase and a glucose or a modified glucose.

120. The method of claim 111 wherein the nucleic acid comprises a segment of isolated genomic DNA.