Methods for Shearing and Tagging DNA for Chromatin Immunoprecipitation and Sequencing

Patent metadata

Field	Value
Publication number	US-2016208323-A1
Application number	US-201414900217-A
Country	US
Kind code	A1
Filing date	Jun 19, 2014
Priority date	Jun 21, 2013
Publication date	Jul 21, 2016
Grant date	—

Abstract

Disclosed are methods for shearing and tagging chromatin DNA. The disclosed methods include contacting chromatin DNA with at least one transposome, that includes a transposase enzyme. The transposon is made up of a first DNA molecule that includes a first transposase recognition site and a second DNA molecule that includes a second transposase recognition site, wherein the transposase integrates the first and second DNA molecules into chromatin DNA. The first and second DNA molecules of the transposon can be disconnected, such that upon integration of the transposon the chromatin bound DNA is sheared and tagged with the first and second DNA molecules, for example to prepare a library of sheared and tagged chromatin DNA fragments.

First claim

1 . A method for shearing and tagging chromatin DNA,
comprising: contacting chromatin DNA, under conditions that permit integration of a transposon into chromatin DNA, with at least one transposome, the transposome comprising: at least one transposase; and a transposon comprising:
a first DNA molecule comprising a first transposase recognition site; and
a second DNA molecule comprising a second transposase recognition site wherein the at least one transposase integrates the first and second DNA molecules into chromatin DNA,

thereby shearing and tagging chromatin DNA with the first and second DNA molecules.

2 . The method of claim 1 , wherein the first and/or second DNA molecule further comprises a barcode, a sequencing adapter, or a universal priming site.

3 . (canceled)

4 . (canceled)

5 . The method of claim 1 , wherein the at least one transposase comprises a Tn5 transposase, a Mu transposase, an IS5 transposase, an IS91 transposase, or a combination thereof.

6 . (canceled)

7 . (canceled)

8 . The method of claim 1 , wherein the least one transposome comprises at least two different transposomes, and wherein the different transposomes integrate different DNA sequences into the chromatin DNA.

9 . The method of claim 8 , wherein the chromatin DNA comprises chromatin DNA from a single cell.

10 . The method of claim 1 , further comprising providing chromatin DNA.

11 . The method of claim 10 , wherein providing chromatin DNA comprises providing cross-linked chromatin, wherein the chromatin is cross-linked to chromatin associated factors.

12 . The method of claim 11 , further comprising cross-linking chromatin to chromatin associated factors to chromatin DNA.

13 . The method of claim 12 , further comprising contacting the chromatin-associated factor cross-linked to the chromatin DNA with a specific binding agent that specifically binds to the chromatin-associated factor.

14 . The method of claim 13 , further comprising releasing the nucleic acid from the chromatin-associated factor.

15 . The method of claim 13 , wherein the specific binding agent is attached to a solid support.

16 . The method of claim 13 , wherein the specific binding agent is an antibody.

17 . The method of claim 1 , further comprising isolating DNA fragments produced.

18 . The method of claim 17 , wherein the DNA fragments are isolated based on size.

19 . The method of claim 18 , further comprising analyzing the isolated nucleic acid fragments.

20 . The method of claim 19 , wherein analyzing the isolated nucleic acid fragments comprises determining the nucleotide sequence.

21 . The method of claim 20 , wherein the nucleotide sequence is determined using sequencing or hybridization techniques with or without amplification.

22 . The method of claim 1 , further comprising increasing the accessibility of closed chromatin to the at least one transposome.

23 . The method of claim 22 , wherein increasing the accessibility of closed chromatin to the at least one transposome, comprises one or more of contacting the chromatin DNA with MNase, contacting the chromatin DNA with a restriction enzyme whose recognition sites are located with high concentration in closed chromatin, minimally shearing the chromatin DNA or exposing the chromatin DNA to high salt conditions.

24 . A method for preparing a library of sheared and tagged chromatin DNA fragments comprising the method of claim 1 .

25 . A chromatin immuno-precipitation tagementation kit, the kit comprising:
a cross-linking agent; a first specific binding agent that binds to a chromatin-associated factor, or is coated with a molecule that binds to the first affinity molecule, to form a first affinity surface, and a transposase; and the transposon comprising:
a first DNA molecule comprising a first transposase recognition site; and
a second DNA molecule comprising a second transposase recognition site,
wherein the transposase integrates the first and second DNA molecules into chromatin DNA.

26 . The kit of claim 25 , wherein the first and/or second DNA molecule further comprises a barcode, a sequencing adaptor, or a universal priming site.

27 . (canceled)

28 . (canceled)

29 . The kit of claim 25 , wherein the transposase is a Tn5 transposase, a Mu transposase, an IS5, or an IS91 transposase.

30 . (canceled)

31 . (canceled)

Assignees

• Broad Inst Inc
• Harvard College
• Gen Hospital Corp Dba Massachusetts General Hospital
• Massachusetts Inst Technology

Inventors

• Bernstein Bradley
• Goren Alon
• Nusbaum Chad
• Ram Oren
• Rotem Assaf
• Tarjan Daniel
• Xing Jeffrey
• Regev Aviv

Classifications

• G01N33/541

  Double or second antibody {, i.e. precipitating antibody} · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title