METHODS AND KITS FOR ANALYZING DNA BINDING MOIETIES ATTACHED TO DNA

• US 20190203270A1
• Filed: 07/24/2016
• Published: 07/04/2019
• Est. Priority Date: 07/24/2016
• Status: Active Application

• Current Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: AMIT, Ido, LARA-ASTIASO, David, GURY, Meital
• Application Number:
  US16/319,499
• Publication Number:
  US 20190203270A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

Methods of analyzing DNA molecules in a cell sample are provided. The DNA molecules have DNA binding moiety signatures which are defined by at least two non-identical DNA binding moieties. Kits for analyzing the DNA are also provided.

Claims

1. A method of analyzing DNA molecules in a cell sample, said DNA molecules having DNA binding moiety signatures which are defined by at least two non-identical DNA binding moieties, the method comprising:

(a) labeling DNA molecules of the cell sample with a label that indexes the identity of at least two of the DNA binding moieties of said signatures so as to generate subpopulations of differentially labeled DNA molecules, each subpopulation being in a separate container, wherein said labeling indexes one DNA binding moiety per DNA molecule;

(b) labeling said differentially labeled molecules with another label which indexes the identity of another DNA binding moiety of said signature; and

(c) analyzing the DNA comprising said first label and said second label.

2. The method of claim 1, wherein said label is a nucleic acid label.

3. The method of claim 1, wherein said labeling of step (a) comprises attaching no more than one label per DNA molecule.

4. The method of claim 1, wherein said labeling comprises end-labeling.

5. The method of claim 1, wherein said labeling of step (b) comprises attaching no more than one label per DNA molecule.

6. The method of claim 1, further comprising repeating step (b) using an additional label prior to step (c).

7. The method of claim 1, wherein neither said first DNA binding moiety nor said second DNA binding moiety bind to more than 50% of the DNA of the sample.

8. The method of claim 1, further comprising pooling said subpopulations to generate a pooled sample of differentially labeled DNA molecules following step (a) and prior to step (b).

9. The method of claim 1, further comprising shearing the DNA of the cell sample prior to step (a).

10. The method of claim 1, wherein said analyzing comprises sequencing said DNA.

11. The method of claim 1, further comprising analyzing said DNA binding moieties following step (b).

12. The method of claim 1, wherein said DNA is no longer than 500 bases.

13. The method of claim 1, wherein said DNA binding moiety is a DNA binding protein.

14. The method of claim 13, wherein said DNA binding protein is a histone.

15. The method of claim 13, wherein said DNA binding protein is a transcription factor.

16. The method of claim 1, wherein said DNA binding moiety is a drug.

17. The method of claim 1, wherein said sample is derived from cells of a single type or line.

18. The method of claim 14, wherein said histone is a post-translationally modified histone.

19. The method of claim 18, wherein said post-translationally modified histone is a methylation or acetylation.

20. The method of claim 19, wherein said post-translationally modified histone is selected from the group consisting of H3K4me1, H3K4me2, H3K4me3 and H3K27ac.

21. A kit for immunoprecipitating a DNA-protein complex comprising:

(i) at least one antibody which specifically binds to a transcription factor;

(ii) at least one antibody which specifically binds to a post-translationally modified histone; and

(iii) a DNA labeling agent.

22. The kit of claim 21, further comprising an antibody for immobilizing at least 50% of the chromatin of a cell.

23. The kit of claim 21, further comprising at least one agent selected from the group consisting of an RNA polymerase, a DNAse and a reverse transcriptase.

24. The kit of claim 21, further comprising a plurality of barcode DNA sequences.

25. The kit of claim 21, further comprising a solid support for immobilizing said at least one antibody.

26. The kit of claim 21, further comprising at least one component selected from the group consisting of a crosslinker, a protease enzyme and a ligase.