DNA microarrays comprising active chromatin elements and comprehensive profiling therewith
AssigneeRexagen Corporation
InventorsJohn A. Stamatoyannapoulos, Michael McArthur
Life of this patent
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Abstract
Arrays, probes and methods are disclosed for the construction and interrogation of DNA arrays containing Active Chromatin Elements, and thereby active genetic regulatory sequences. Further methods are disclosed for interrogation of such arrays in order to reveal the pattern of genetic regulatory activity within any given cell or tissue type or associated with any particular genetic locus under a variety of conditions.
Claims126
A nucleic acid array comprising a plurality of active chromatin elements.
A method for determining the active chromatin element profile of nuclear chromatin of a cell comprising:
- treating a portion of said chromatin with an agent that preferentially modifies DNA at hypersensitive sites to form a first set of nucleic acids;
- treating another portion of said chromatin with another agent that non-preferentially modifies DNA to form a second set of nucleic acid;
- and
- comparing the first and second sets to obtain said active chromatin element profile.
A method for identifying a profile of DNA regulatory elements in a eukaryotic cell comprising:
- treating said cell with an agent that modifies DNA of said cell at DNA hypersensitive sites;
- and
- identifying the DNA hypersensitive sites from said reaction with the agent, wherein the nucleotide sequences of said DNA hypersensitive sites and the locations thereof in the DNA of said type of cells constitute a profile of DNA regulatory elements in said type of cells.
A method for producing a profile of DNA regulatory elements in eukaryotic cells, comprising:
- treating said cells with an agent that modifies eukaryotic DNA at DNA hypersensitive sites;
- identifying the DNA hypersensitive sites from said reaction with said agent wherein the nucleotide sequences of said DNA hypersensitive sites and the locations thereof in the DNA of said type of cells constitute a profile of DNA regulatory elements in said type of cells;
- and
- isolating the nucleotide sequences of said hypersensitive sites.
A method for detecting DNA regulatory elements in eukaryotic cells comprising:
- a) isolating mRNAs from said cells, converting said mRNA's to cDNA and probing an array to generate a profile;
- b) isolating active regulatory elements from said cells and probing an array to generate a profile, and
- c) comparing the profile from the cDNA probe with the profile from the active regulatory elements probe to correlate regulatory element activity with gene activity.
A sequence library of active chromatin elements encoding fragments suitable for preparing a profile to determine the regulatory status of a eukaryotic cell sample.
A library of active chromatin element primers, prepared by obtaining a library of active chromatin element fragments and determining sequences outside the active chromatin element fragments suitable for cloning the active chromatin element fragments.
A method for profiling active chromatin elements from a sample that contains nucleic acid, comprising:
- a) obtaining one or more purified or labeled active chromatin elements from the sample;
- b) contacting the active chromatin elements from step a) with a DNA microarray containing DNA species in separate locations that match sites of the genome;
- and
- c) detecting binding between the active chromatic elements and sites of the microarray.
A method of discerning at least one set of co-regulated genes in cells of a eukaryotic organism, comprising:
- obtaining a first profile for binding between active chromatic elements of the tissue under controlled culture conditions;
- obtaining a second profile for binding between active chromatic elements of the tissue under conditions where a known regulator of at least one of the genes is altered with respect to the controlled culture conditions;
- and
- comparing the first profile with the second profile from b) to determine which genetic elements are effected by the alteration of the known regulator.
A method for profiling active chromatin elements from a sample that contains nucleic acid, comprising:
- a) obtaining one or more purified active chromatin elements from the sample and label them;
- b) contacting the labeled active chromatin elements from step a) with a DNA microarray containing DNA species in separate locations that match putative or verified regulatory elements;
- and
- c) detecting binding between the active chromatic elements and sites of the microarray.
A method for profiling active chromatin elements from a sample that contains nucleic acid, comprising:
- a) obtaining multiple active chromatin elements from the sample and label them with a first label;
- b) obtaining multiple genomic DNA fragments from the sample and label them with a second label;
- c) hybridizing the elements from a) and the fragments from b) with a DNA microarray containing DNA species in separate locations that match putative or verified regulatory elements;
- and
- d) determining the ratio of signals from the first and second labels within the array.
A method for profiling differential regulatory element activation from two populations that contain nucleic acid, comprising:
- a) obtaining multiple active chromatin elements from the first population and labeling them with a first label;
- b) obtaining multiple active chromatin elements from the second population and labeling them with a second label;
- c) hybridizing the elements from a) and the fragments from b) with a DNA microarray containing DNA species in separate locations that match putative or verified regulatory elements;
- and
- d) determining the ratio of signals from the first and second labels within the array.
A method for correlating regulatory element activation with gene expression from a sample that contains nucleic acid, comprising:
- a) obtaining multiple active chromatin elements from the sample and profiling them on a DNA microarray containing DNA species in separate locations that match putative or verified regulatory elements;
- b) isolating RNA from the sample and converting to cDNA;
- c) profiling the cDNA on a DNA microarray containing DNA species in separate locations that match putative or verified regulatory elements;
- and
- d) correlate the profile results from a) and c) with gene activity using informatics software.
A method of identifying an ACE profile associated with a disease state, comprising;
- a) obtaining a first profile or set of profiles for binding between active chromatin elements of a tissue, said first profile or set of profiles being representative of a normal healthy condition;
- b) obtaining a second profile or set of profiles for binding between active chromatin elements of a tissue, said second profile or set of profiles being representative of a disease condition;
- and
- c) comparing the first profile or set of profiles with the second profile or set of profiles to identify alterations in the activity of one or more ACE elements in the disease condition relative to the normal condition.
A method for isolating ACE sequences in a eukaryotic cell comprising:
- a) preparing nuclei from a biological sample;
- b) treating the nuclei to form cross-linked chromatin-protein complexes;
- c) treating the cross-linked chromatin-protein complexes to reduce the size of the DNA sequences associated with the complexes;
- d) capturing the chromatin-protein complexes;
- and
- e) isolating DNA sequences associated with the chromatin-protein complexes.
A method of preparing fixed length direct monotagged nucleic acids comprising:
- a) treating genomic DNA with an agent that cleaves DNA;
- b) ligating the treated genomic DNA with a blunt or T-tailed linker containing a type IIs restriction endonuclease restriction site;
- and
- c) treating the ligated DNA with a type IIs restriction enzyme.
A method of preparing fixed length indirect monotagged nucleic acids comprising:
- a) treating genomic DNA with an agent that cleaves DNA;
- b) capturing the treated genomic DNA;
- c) treating the captured genomic DNA with a restriction enzyme;
- d) ligating the genomic DNA of step c) with a linker comprising a type IIs restriction enzyme site;
- and
- e) treating the ligated DNA with a type II restriction enzyme.
Dependencies are inferred from the claim text.