Methods and compositions for discrimination between cytosine and modifications thereof and for methylome analysis

• US 9,121,061 B2
• Filed: 03/14/2013
• Issued: 09/01/2015
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Vaisvila, Romualdas, Johnson, Heidi Erika, Vainauskas, Saulius, Davis, Theodore B.
• Primary Examiner(s): Prouty, Rebecca
• Assistant Examiner(s): EKSTROM, RICHARD C
• Application Number:
  US13/827,885
• Publication Number:
  US 20130244237A1
• Time in Patent Office: 901 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• 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 2521/539 Deaminase
• CPC Class Codes: C12Y 114/11 with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 305/04005 Cytidine deaminase (3.5.4.5)

Abstract

Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.

Claims

1. A method for differentiating unmethylated cytosine (C) and 5-methylcytosine (5-mC) from 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-CaC) or 5-glycosylated hydroxymethylcytosine (5-ghmC), comprising:

(a) reacting a polynucleotide optionally containing C, 5-mC, 5-hmC, 5-fC, 5-CaC and/or 5-ghmC, with a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO;

2, wherein 5-mC is converted to a thymine (T) and C is converted to a uracil (U); and

(b) amplifying or cleaving the polynucleotide to identify the location of at least one converted nucleotide in the polynucleotide.

2. A method according to claim 1, further comprising differentiating 5-hydroxymethylcytosine (5-hmC), from unmethylated cytosine (C) by reacting the polynucleotide prior to (a) with an oxygenase so as to generate a sequence wherein only C is altered to uracil (U).

3. A method according to claim 2, further comprising sequencing the polynucleotide in which unmethylated cytosine (C) is converted to uracil (U) and sequencing the polynucleotide obtained in (a) where C is converted to U and 5-methylcytosine (5-mC) is converted to thymine (T) and comparing the sequences to characterize 5-mC in the polynucleotide.

4. A method according to claim 1, further comprising:

constructing a methylome map.

5. A method according to claim 1, wherein the polynucleotide in (a) is single-stranded.

6. A method according to claim 1, further comprising, comparing the sequence of the deaminated polynucleotide from (a) with the sequence of the untreated polynucleotide.

7. A method according to claim 1, wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

8. A method according to claim 1, wherein cleaving the polynucleotide further comprises cleaving the polynucleotide with a glycosylase and endonuclease at a uracil (U) or cleaving the polynucleotide after DNA amplification, with a restriction endonuclease that recognizes a site after conversion of unmethylated cytosine (C) to thymine (T) in the polynucleotide.

22. The method of claim 7, wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:

2.

9. A method for differentiating unmethylated cytosine (C) from 5-methylcytosine (5-mC), 5-hydroxymethyl cytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-CaC) and/or 5-glycosylated hydroxymethylcytosine (5-ghmC) in a polynucleotide, comprising:

(a) reacting a first sample of the polynucleotide optionally containing C, 5-mC, 5-hmC, 5-fC, 5-CaC and/or 5-ghmC, with an oxygenase followed by a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO;

2, thereby converting C to uracil (U) and converting 5-mC to 5-hmC and 5-CaC; and

(b) amplifying or cleaving the polynucleotide to identify the location of the U in the polynucleotide.

10. A method according to claim 9, further comprising:

reacting a second sample of the polynucleotide with a cytidine deaminase in the absence of an oxygenase for converting an unmethylated cytosine (C) to uracil (U) and 5-methylcytosine (5-mC) to thymine (T).

11. A method according to claim 9, wherein the polynucleotide from step (a) is single-stranded.

12. A method according to claim 10, further comprising:

sequencing the first sample of the polynucleotide after the reaction with the oxygenase and cytidine deaminase to generate a first sequence;

sequencing the second sample of the polynucleotide after a reaction with cytidine deaminase but not with the oxygenase to generate a second sequence;

optionally sequencing a third sample of the polynucleotide absent a reaction with the cytidine deaminase or the oxygenase; and

comparing the first sample sequence and the second sample sequence and optionally the third sample sequence to detect and differentiate unmethylated cytosine (C) and 5-methylcytosine (5-mC).

13. A method according to claim 9, wherein the oxygenase is a methylpyrimidine oxygenase or a 5-methylcytosine oxygenase.

14. A method according to claim 13, wherein the 5-methylcytosine oxygenase is TET1.

15. A method according to claim 13, wherein the methylpyrimidine oxygenase is a mYOX.

16. A method according to claim 9, wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

23. The method of claim 16, wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:

2.

17. A method for differentiating a 5-methylcytosine (5-mC) from an unmethylated cytosine (C), comprising:

reacting a first sample of a polynucleotide with sodium bisulfite sequencing reagents followed by a cytidine deaminase having an amino acid sequence that is at least 90% identical to SEQ ID NO;

2, in the absence of an oxygenase, thereby converting 5-mC to thymine (T) and converting C to uracil (U); and

reacting a second sample of the polynucleotide with sodium bisulfite sequencing reagents without subsequent exposure to a cytidine deaminase, thereby converting C to U while retaining 5-mC as 5-mC.

18. A method according to claim 17, further comprising amplifying the first and second samples of the polynucleotides for sequencing to identify the presence and location of at least one converted nucleotide in the polynucleotides.

19. A method according to claim 17, wherein the cytidine deaminase has an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

20. A method according to claim 17, wherein the method is performed at a temperature less than 60°

C.

21. A method according to claim 17, wherein the polynucleotide has a length of greater than 1 Kb.

24. The method of claim 19, wherein the cytidine deaminase has a mutation at one or more positions corresponding to positions 23, 25, 29, 45, 69, 104 and 123 in SEQ ID NO:

2.