Compositions and Methods for Analyzing Modified Nucleotides

• US 20180171397A1
• Filed: 02/09/2018
• Published: 06/21/2018
• Est. Priority Date: 10/30/2015
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Vaisvila, Romualdas, Davis, Theodore B., Guan, Shengxi, Sun, Zhiyi, Ettwiller, Laurence, Saleh, Lana
• Granted Patent: US 10,260,088 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2521/539 Deaminase
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• 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 305/04005 Cytidine deaminase (3.5.4.5)

Abstract

Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map ^5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map ^5hmC in a DNA.

Claims

1. A method, comprising:

a. treating an aliquot of a nucleic acid sample with a methylcytosine dioxygenase and optionally a glucosyltransferase in a reaction mix to produce a reaction product in which the modified cytosines (Cs) are oxidized and optionally the 5-hydroxymethylcytosines (^5hmCs) are glucosylated; and

b. treating the reaction product of (a) with cytidine deaminase to form a second reaction product in which substantially all unmodified Cs are converted to uracil (U).

2. The method according to claim 1, further comprising:

treating a second aliquot of the nucleic acid sample with glucosyltransferase in the absence of a dioxygenase and subsequently with cytidine deaminase to produce a third reaction product in which substantially all the 5-hydroxymethylcytosines (^5hmCs) in the aliquot are glucosylated, substantially all the unmodified Cs are converted to Us and methylcytosines (^5mCs) are converted to Thymine (Ts).

3. The method according to claim 1, wherein the nucleic acid sample is a library of DNA fragments wherein the DNA fragments are ligated to nucleic acid adaptors in which cytosine in the adaptors is replaced by pyrollo-dC.

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

sequencing the second reaction product or amplification product thereof to determine which cytosines (Cs) are methylated and which are unmethylated.

5. The method according to claim 2, further comprising sequencing the third reaction product or an amplification product thereof to determine which cytosines are hydroxymethylated.

6. The method according to claim 1, further comprising obtaining a reference sequence by sequencing a third aliquot of the nucleic acid sample not reacted with the dioxygenase, glucosyltransferase or deaminase, or obtaining a reference sequence from a database of sequences.

7. The method according to claim 5, further comprising:

comparing the sequences obtained from the second reaction product and the reference sequence and optionally the third reaction product to determine which cytosines (Cs) in the nucleic acid sample are unmodified and optionally which of the modified cytosine are methylated or hydroxymethylated

8. The method of claim 1, wherein the nucleic acid sample contains fragments that are linked, in cis, to a gene in a transcriptionally active or inactive state whereas the reference sequence is the same fragment that is linked, in cis, to the same gene in an opposite transcriptionally active or inactive state;

and determining an altered pattern of cytosine methylation associated with transcriptional activity.

9. The method of claim 1, wherein the altered pattern of cytosine methylation associated with transcriptional activity of the gene in an ex vivo cell or tissue is correlated with a disease or condition.

10. The method of claim 1, wherein the method comprises comparing (i) the pattern of cytosine methylation for a nucleic acid fragment from a mammalian subject that has a disease or condition with (ii) the pattern of cytosine methylation in the same nucleic acid fragment from a mammalian subject that does not have the disease or condition.

11. The method of claim 1, wherein the method comprises comparing (i) the pattern of cytosine methylation for a nucleic acid fragment from a mammalian subject undergoing a treatment with (ii) the pattern of cytosine methylation in the same intact nucleic acid fragment from a mammalian subject that has not been treated with the agent for detecting differences.

12. The method of claim 1, wherein the methylcytosine dioxygenase and glucosyltransferase are combined within a single reagent for adding to the reaction together.

13. The method of claim 1, wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:

1 and comprises the amino acid sequence of SEQ ID NO;

2.

14. The method of claim 1, wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:

3.

15. The method of claim 1, wherein the cytidine deaminase is APOBEC3A.

16. A kit comprising:

(a) a eukaryotic or prokaryotic deaminase; and

(b) a bacteriophage dioxygenase.

17. A kit according to claim 16, further comprising a glucosyl transferase.

18. A kit according to claim 16, further comprising adaptor oligonucleotides in which all the cytosines (Cs) have been replaced with pyrollo-dC.

19. A kit according to claim 16, further comprising a dU bypass polymerase.

20. A kit according to claim 17, wherein the glucosyltransferase and the dioxygenase are combined in a single tube or contained in separate tubes.

21. The kit of claim 17, wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:

1 and comprises the amino acid sequence of SEQ ID NO;

2.

22. The kit of claim 17, wherein the methylcytosine dioxygenase has an amino acid sequence that is at least 90% identical to SEQ ID NO:

3.

23. The kit of claim 16, wherein the deaminase is APOBEC3A.

24. A method, comprising:

treating an aliquot of a nucleic acid sample with glucosyltransferase in the absence of dioxygenase and subsequently with cytidine deaminase to produce a reaction product in which substantially all the 5-hydroxymethylcytosines (^5hmCs) are glucosylated and substantially all the Cs are converted to Us and substantially all the 5-methylcytosines (^5mCs) are converted to Ts.

25. The method according to claim 24, comprising:

determining the location of 5-hydroxymethylcytosine (^5hmC) in the sample.