Detection and quantification of hydroxymethylated nucleotides in a polynucleotide preparation

• US 9,034,597 B2
• Filed: 08/25/2010
• Issued: 05/19/2015
• Est. Priority Date: 08/25/2009
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Bitinaite, Jurate, Vaisvila, Romualdas, Pradhan, Sriharsa, Zheng, Yu, Roberts, Richard J., Cohen-Karni, Devora, Noren, Christopher, Raleigh, Elisabeth A., Wilson, Geoffrey, Chin, Hang-Gyeong
• Primary Examiner(s): Switzer, Juliet
• Assistant Examiner(s): Hammell, Neil P
• Application Number:
  US13/392,286
• Publication Number:
  US 20120156677A1
• Time in Patent Office: 1,728 Days
• Field of Search: None
• US Class Current: 435/61
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

Methods and compositions are described for detecting hydroxymethylated nucleotides (hmNs) in a polynucleotide preparation with a view to mapping the location of hmNs in a genome, quantifying the occurrence of hmNs at selected loci and correlating the occurrence of hmNs with gene expression and phenotypic traits. Embodiments describe the use of modifying enzymes together with site-specific endonucleases to detect the hmNs.

Claims

1. A method of quantifying the amount of a hydroxymethylated nucleotide (hmN) in a polynucleotide preparation, comprising:

(a) obtaining a preparation of polynucleotides;

(b) modifying any hmN in the polynucleotides of a first portion of the preparation to produce modified hmN (mhmN), wherein the modifying is done using a DNA glucosyltransferase; and

(c) quantifying the amount of hmN in the preparation by;

(i) reacting the polynucleotides from (b) with a site-specific endonuclease selected from BccI, BciVI, BspHI, BspQI, BstEII, BstNI, BstYI, BstCI, CviAII, CviQI, DpnI, EcoRI, HinfI, Hpy188I, Hpy188III, MboII, MfeI, MlyI, NsiI, RsaI, ScaI, SfcI, SmlI, Tsp45I, XbaI, BsaWI, BsoBI, BspEI, BssI, BtgZI, EciI, MspI, NmeAII, PspXI, TliI, XhoI, and XmaI; and

(ii) comparing the amount of an uncleaved polynucleotide that would otherwise be cleaved but for the modified hmN with the amount of uncleaved polynucleotide produced by digesting a control sample by the site-specific endonuclease, wherein the control sample is a second portion of the preparation that has not been modified to produce mhmN.

2. The method of claim 1, wherein the N, mN, hmN and mhmN are C, mC, hmC and mhmC respectively.

3. The method of claim 1, wherein the site-specific endonuclease is also capable of cleaving a polynucleotide at the specific recognition site comprising mN.

4. The method of claim 1, wherein the site-specific endonuclease is also capable of cleaving a polynucleotide at the specific recognition site when the nucleotide is a mN and when the nucleotide is a N.

5. The method of claim 1, further comprising distinguishing between an unmethylated nucleotide (N) and a methylated nucleotide (mN) in the polynucleotide preparation.

6. The method of claim 5, comprising reacting a control sample of the polynucleotide preparation with a second site-specific endonuclease, wherein the second site-specific endonuclease is capable of cleaving a polynucleotide at a specific recognition site containing the nucleotide when the nucleotide is a N, but not when the nucleotide is a mN or a hmN, and detecting a cleaved polynucleotide;

and thereby determining that the nucleotide in the polynucleotide preparation is a N.

7. The method of claim 1, wherein an adapter is ligated to the polynucleotides in the sample for amplifying or sequencing an uncleaved polynucleotide.

8. The method of claim 1, wherein the method further comprises mapping an hmN detected in c) to a genomic locus.

9. The method of claim 1, wherein the polynucleotide preparation is derived from a cell, tissue or organism and wherein (c) comprises identifying an hmN at a predetermined locus in a genome for the polynucleotide in the preparation.

10. The method of claim 9, wherein the method comprises determining an amount of the hmN in the predetermined locus in the genome from a cell, tissue or organism.

11. The method of claim 1, wherein the method comprises comparing the amount of hmN in polynucleotides in a first polynucleotide preparation to the amount of hmN in polynucleotides in a second polynucleotide preparation.

12. The method of claim 1, further comprising correlating with a phenotypic trait, a difference in the amount of the hmN at a predetermined locus in a first polynucleotide in a first polynucleotide preparation and in a second polynucleotide in a second polynucleotide preparation.

13. The method of claim 1, wherein further comprising recording in a computer-readable form the amount of hmN measured in (c).

14. A method of quantifying the amount of a hydroxymethylated cytosine (hmC) in a polynucleotide preparation, comprising:

(a) obtaining a preparation of polynucleotides;

(b) modifying any hmC in the polynucleotides of a first portion of the preparation to produce modified hmC (mhmC), wherein the modifying is done using a DNA glucosyltransferase; and

(c) quantifying the amount of hmC in the preparation by;

(i) reacting the polynucleotides from (b) with MspI; and

(ii) comparing the amount of an uncleaved polynucleotide that would otherwise be cleaved but for the mhmC with the amount of uncleaved polynucleotide produced by digesting a control sample by MspI, wherein the control sample is a second portion of the preparation that has not been modified to produce mhmC.