Methods for studying nucleic acids
View Patent ↗The present invention provides a novel method for preparing a sequencing library and studying molecular interactions involving a nucleic acid. In particular, the invention relates to a method for preparing a sequencing library, the method comprising the addition of an agent binding to chromatin to a sample comprising a nucleic acid; isolating chromatin bound by said agent; addition of transposase to the isolated chromatin; isolating nucleic acid from chromatin; and obtaining a sequencing library. Moreover, the present invention relates to a method for mapping of molecular interactions involving a nucleic acid, the method comprising the addition of an agent binding to chromatin to a sample comprising a nucleic acid; isolating chromatin bound by said agent; addition of transposase to the isolated chromatin; isolating nucleic acid from chromatin; amplification of nucleic acid; sequencing of amplified nucleic acid; and identifying molecular interactions.
1. A method for isolating nucleic acid from chromatin, the method comprising:
(a) adding an agent binding to chromatin to a sample comprising a nucleic acid;
(b) isolating the chromatin bound by said agent;
(c) adding a transposase to the isolated chromatin of step (b); and
(d) isolating the nucleic acid from the chromatin of step (c).
2. The method of claim 1 , wherein the method further comprises generating a sequence library from the isolated nucleic acid of (d).
3. The method of claim 2 , wherein the agent is an antibody.
4. The method of claim 1 , wherein the method further comprises:
(e) amplifying the isolated nucleic acid of (d);
(f) sequencing the amplified nucleic acid of (e); and
(g) identifying molecular interactions of the nucleic acid.
5. The method of claim 4 , wherein the agent is an antibody.
6. The method of claim 1 , wherein the nucleic acid is DNA.
7. The method of claim 1 , wherein the sample comprising a nucleic acid has been prepared by
(i) cultivating and harvesting cells;
(ii) fixing cells;
(iii) lysing cells and thereby obtaining a first sample comprising a nucleic acid; and
(iv) sonicating the first sample and thereby obtaining a second sample comprising a nucleic acid, wherein said second sample is to be used in the method of claim 1 .
8. The method of claim 7 , wherein the method further comprises a step of reversing cross-links introduced during fixing cells of step (ii).
9. The method of claim 7 , wherein the cells comprise nucleic acid-protein complexes.
10. The method of claim 7 , wherein the cells are human cells, animal cells, bacterial cells, yeast cells, archaeal cells, plant cells or viruses.
11. The method of claim 10 , wherein the human or animal cells are diseased cells or non-diseased cells or cells derived from diseased or non-diseased tissue.
12. The method of claim 10 , wherein the human or animal cells are cancer cells, immune cells, blood cells or stem cells.
13. The method of claim 12 , wherein the cancer cells are derived from a solid cancer or blood cancer.
14. The method of claim 13 , wherein the blood cancer is leukemia.
15. The method of claim 13 , wherein the solid cancer is a tumour.
16. The method of claim 10 , wherein the animal cells are derived from an animal belonging to a rare species, endangered species and/or is a model organism.
17. The method of claim 10 , wherein the cells are embryonic cells.
18. The method of claim 7 , wherein step (ii) comprises the addition of a chemical substance and/or physical means.
19. The method of claim 18 , wherein the chemical substance is formaldehyde or paraformaldehyde.
20. The method of claim 18 , wherein the physical means comprise UV-light or laser.
21. The method of claim 7 , wherein step (iv) comprises sonication until most of the nucleic acid fragments are 20-5000, preferably 200-300, base pairs long.
22. The method of claim 1 , wherein the agent binding to chromatin is a chemical substance.
23. The method of claim 22 , wherein the chemical substance is a drug or a tool compound.
24. The method of claim 23 , wherein the chemical substance is biotinylated.
25. The method of claim 1 , wherein the agent is an antibody that specifically binds to histones, transcription factors or proteins binding to histones and/or transcription factors.
26. The method of claim 25 , wherein the proteins binding to histones and/or transcription factors are nucleic acid remodeling proteins or chromatin modifying enzymes.
27. The method of claim 25 , wherein the histone is H3.3, H2A.Z, CENP-A, H3.2, H3.3A, H3.3B, H4 or H3.1.
28. The method of claim 25 , wherein the histone is a modified histone, wherein the modification is methylation, acetylation, propionylation, butyrylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, succinylation and/or ribosylation.
29. The method of claim 28 , wherein the modified histone is H3K4me1/2/3, H2BK5me1, H3K27me1/2/3, H3K9me1/2/3, H4K20me1, H3K79me1, H3K36me3, H2AK5ac, H2AK9ac, H2BK5ac, H2BK12ac, H2BK20ac, H2BK120ac, H3K4ac, H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K36ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac, H4K91ac, H2Aub or H2Bub.
30. The method of claim 1 , wherein the transposase comprises random DNA sequence tags or defined DNA sequence tags.
31. The method of claim 30 , wherein the transposase is a Tn5 transposase.