SIMULTANEOUS SINGLE-MOLECULE EPIGENETIC IMAGING OF DNA METHYLATION AND HYDROXYMETHYLATION

• US 20170298422A1
• Filed: 03/27/2017
• Published: 10/19/2017
• Est. Priority Date: 04/18/2016
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Song, Chunxiao, Quake, Stephen R., Brunger, Axel, Diao, Jiajie
• Application Number:
  US15/470,635
• Publication Number:
  US 20170298422A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Provided herein is a method for analyzing genomic DNA. In some embodiments, the method may comprise labeling a genomic sample by adding a capture tag to the ends of the DNA molecules in the sample and labeling molecules that comprise hydroxymethylcytosine with a first fluorophore, immobilizing the labeled DNA molecules on a support, and imaging individual molecules of hydroxymethylated genomic DNA on the support.

Claims

1. A method for analyzing genomic DNA, comprising:

(a) labeling a sample comprising the genomic DNA by;

(i) adding a capture tag to the ends of the DNA molecules in the sample; and

(ii) labeling molecules that comprise hydroxymethylcytosine with a first fluorophore;

(b) immobilizing the DNA molecules labeled made in step (a) on a support; and

(c) imaging individual molecules of hydroxymethylated DNA on the support.

2. The method of claim 1, further comprising:

(d) counting the number of individual molecules labeled with the first fluorophore, thereby determining the number of hydryoxymethylated DNA molecules in the sample.

3. The method of claim 1, wherein the first fluorophore of step (a)(ii) is added by incubating DNA molecules with a DNA β

-glucosyltransferase and UDP glucose modified with a chemoselective group, thereby covalently labeling the hydroxymethylated DNA molecules with the chemoselective group, and linking the first fluorophore to the chemoselectively-modified DNA via a cycloaddition reaction.

4. The method of claim 1, wherein step (a)(i) further comprises adding a second fluorophore to the ends of the DNA molecules in the sample.

5. The method of claim 1, wherein the

step (a) further comprises:

after step (ii), (iii) labeling molecules that comprise methylcytosine with a second fluorophore; and

step (c) further comprises imaging individual molecules of methylated DNA on the support.

6. The method of claim 5, further comprising:

(d) counting;

(i) the number of individual molecules labeled with the first fluorophore and (ii) the number of individual molecules labeled with the second fluorophore.

7. The method of claim 6, further comprising

(e) calculating the relative amounts of hydroxymethylated DNA and methylated DNA in the sample.

8. The method of claim 5, wherein the molecules that comprise methylcytosine are labeled with the second fluorophore by:

incubating the product of step (a)(ii) with a methylcytosine dioxygenase, thereby converting methylcytosine into hydroxymethylcytosine;

incubating the methylcytosine dioxygenase-treated DNA with a DNA β

-glucosyltransferase and UDP glucose modified with a chemoselective group, thereby covalently labeling the hydroxymethylated DNA molecules with the chemoselective group, and

linking the second fluorophore to the chemoselectively-modified DNA via a cycloaddition reaction.

9. The method of claim 1, wherein:

step (a) further comprises;

iii. labeling molecules that comprise methylcytosine with a second fluorophore; and

step (c) comprises imaging individual molecules of genomic DNA by detecting a FRET (fluorescence resonance energy transfer) signal emanating from the first or second fluorophores of (a)(ii) or (a)(iii);

wherein a FRET signal indicates that a molecule has a hydroxymethylcytosine and a methylcytosine that are proximal to one another.

10. The method of claim 9, wherein the method comprises determining if the molecule has a proximal hydroxymethylcytosine and methylcytosine on the same strand.

11. The method of claim 9, wherein the method comprises determining if the molecule has a proximal hydroxymethylcytosine and methylcytosine on different strands.

12. The method of claim 1, wherein the capture tag of step (a)(i) is added by incubating the sample with a terminal transferase and a biotinylated nucleotide.

13. The method of claim 1, wherein the imaging is done by single-molecule total internal reflection fluorescence (TIRF) microscopy.

14. The method of claim 1, wherein the genomic DNA comprises eukaryotic DNA.

15. The method of claim 1, wherein the genomic sample comprises fragmented genomic DNA.

16. The method of claim 1, wherein the genomic sample comprises cfDNA.

17. A method of sample analysis, comprising:

(a) analyzing, using the method of any prior claim;

(i) hydroxymethylated DNA and, optionally, the amount of methylated DNA in a first sample and (i) hydroxymethylated DNA and, optionally, the amount of methylated DNA in second sample of cfDNA; and

(b) comparing the results obtained in step (a) to determine if there is a difference in hydroxymethylation or methylation between the samples.

18. The method of claim 17, wherein at least one of the samples is a clinical sample.

19. A method for labeling cell-free DNA (cfDNA), comprising:

attaching labels to DNA molecules that comprise one or more hydroxymethylcytosine and methylcytosine nucleotides in a cfDNA sample, wherein the hydroxymethylcytosine nucleotides are labeled with a first label and the methylcytosine nucleotides are labeled with a second label that is different to the first label, to produce a labeled sample.

20. The method of claim 19, wherein the first and second labels are distinguishable fluorophores.

21. The method of claim 19, wherein the first and second labels are different capture tags.

22. The method of claim 19, wherein the method further comprises analyzing the labeled sample.

23. A method for analyzing genomic DNA molecules, comprising:

(a) labeling a sample comprising the genomic DNA by;

(i) adding a capture tag to the ends of the DNA molecules; and

(ii) attaching labels to DNA molecules that comprise one or more hydroxymethylcytosine and methylcytosine nucleotides, wherein the hydroxymethylcytosine nucleotides are labeled with a first fluorophore and the methylcytosine nucleotides are labeled with a second fluorophore, wherein the first and second fluorophores are capable of generating a FRET signal, to produce labeled genomic DNA; and

(b) detecting a FRET signal from an individual molecule of the labeled genomic DNA, wherein the FRET signal is indicative of the proximity between one or more of the hydroxymethylcytosine and methylcytosine nucleotides in the genomic DNA.

24. The method of claim 23, wherein the hydroxymethylcytosine nucleotides are labeled with a FRET donor and the methylcytosine nucleotides are labeled with FRET acceptor.

25. The method of claim 23, wherein the hydroxymethylcytosine nucleotides are labeled with a FRET acceptor and the methylcytosine nucleotides are labeled with FRET donor.