Functional arrays for high throughput characterization of gene expression regulatory elements
First Claim
1. A library of expression constructs, each member of the library comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, wherein:
- (a) the library has a diversity of at least 50 different nucleic acid segments;
(b) each nucleic acid segment and is naturally linked in the genome with a sequence expressed as a cDNA; and
(c) the average length of the nucleic acid segments in the library is at least 600 nucleotides.
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Abstract
The present invention provides compositions, kits, assemblies, libraries, arrays, and high throughput methods for large scale structural and functional characterization of gene expression regulatory elements in a genome of an organism, especially in a human genome. In one aspect of the invention, an array of expression constructs is provided, each of the expression constructs comprising: a nucleic acid segment operably linked with a reporter sequence in an expression vector such that expression of the reporter sequence is under the transcriptional control of the nucleic acid segment, the nucleic acid segment varying in the library and having a diversity of at least 50. The nucleic acid segments can be a large library of gene expression regulatory elements such as transcriptional promoters. The present invention can have a wide variety of applications such as in personalized medicine, pharmacogenomics, and correlation of polymorphisms with phenotypic traits.
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Citations
71 Claims
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1. A library of expression constructs, each member of the library comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, wherein:
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(a) the library has a diversity of at least 50 different nucleic acid segments;
(b) each nucleic acid segment and is naturally linked in the genome with a sequence expressed as a cDNA; and
(c) the average length of the nucleic acid segments in the library is at least 600 nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 71)
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18. A library of isolated nucleic acid molecules, each member of the library comprising a different, pre-determined nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, wherein:
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(a) the library has a diversity of at least 50 different nucleic acid segments;
(b) each nucleic acid segment is naturally linked in the genome with a sequence expressed as a cDNA; and
(c) the average length of the nucleic acid segments in the library is at least 600 nucleotides.
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19. A library of recombinant nucleic acid molecules, each member of the library comprising a different, determined nucleic acid segment from a genome linked with a heterologous nucleic acid molecule, wherein the segment comprises transcription regulatory sequences, wherein:
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(a) the library has a diversity of at least 50 different nucleic acid segments;
(b) each nucleic acid segment is naturally linked in the genome with a sequence expressed as a cDNA; and
(c) the average length of the nucleic acid segments in the library is at least 600 nucleotides. - View Dependent Claims (20, 21)
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22. A library of cells, wherein each cell in the library of cells comprises a different member of a library of expression constructs, wherein each member of the library of expression constructs comprises a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, wherein:
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(a) the library has a diversity of at least 50 different nucleic acid segments;
(b) each nucleic acid segment is naturally linked in the genome with a sequence expressed as a cDNA; and
(c) the average length of the nucleic acid segments in the library is at least 600 nucleotides. - View Dependent Claims (23, 24)
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25. A collection of cells comprising within the cells a library of expression constructs, each member of the library of expression constructs comprising:
- a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a different heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences.
- View Dependent Claims (26, 27)
- 28. A device comprising at least one plate comprising a plurality of wells, each well containing a different member of a library of expression constructs, each expression construct comprising a different, nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, and wherein each member has a known location among the wells.
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- 35. A device comprising at least one plate comprising a plurality of wells, each well containing a different member of the library of cells, wherein each cell in the library of cells comprises a different member of the library of expression constructs, each expression construct comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences and wherein each member of the library of cells has a known location among the wells.
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37. A kit for characterizing a biological function of a target gene expression regulatory element, comprising:
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(a) a device comprising at least one plate comprising a plurality of wells, each well containing a different member of the library of expression constructs, each expression construct comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, and wherein each member has a known location among the wells; and
(b) reporter assay substrates. - View Dependent Claims (38)
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- 39. A device comprising a solid substrate comprising a surface and nucleic acid molecules immobilized to the surface, each at a different known location, wherein each molecule comprises a nucleotide sequence of at least 10 nucleotides from a genomic segment comprising transcription regulatory sequences and the device comprises transcription regulatory sequences from at least 50 different genomic segments.
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47. A method comprising:
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(a) providing a device comprising at least one plate comprising a plurality of wells, each well containing a different member of a library of cells, wherein each cell in the library of cells comprises a different member of the library of expression constructs, each expression construct comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences and wherein each member of the library of cells has a known location among the wells;
(b) culturing the cells; and
(c) measuring the level of expression of the reporter sequence in each well. - View Dependent Claims (58, 59, 60, 61, 62, 63)
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53. A method comprising:
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(a) providing a first device and second device, each device comprising at least one plate comprising a plurality of wells, each well containing a different member of a library of cells, wherein each cell in the library of cells comprises a different member of the library of expression constructs, each expression construct comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, wherein each member of the library of cells has a known location among the wells and wherein the first and second devices comprise cells of the same type and the library of expression constructs is the same in the first and second devices;
(b) culturing the cells of the first and second devices under different culture conditions;
(c) measuring the level of expression of the reporter sequence in each well; and
(d) comparing the level of expression of the reporter sequence to each transcription regulatory sequence between the first cell type and the second cell type. - View Dependent Claims (54)
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55. A method comprising:
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(a) providing a first device and second device, each device comprising at least one plate comprising a plurality of wells, each well containing a different member of a library of cells, wherein each cell in the library of cells comprises a different member of the library of expression constructs, each expression construct comprising a different nucleic acid segment from a genome, wherein the segment comprises transcription regulatory sequences, operably linked with a heterologous reporter sequence in an expression vector such that expression of the reporter sequence is under transcriptional control of the transcription regulatory sequences, wherein each member of the library of cells has a known location among the wells and wherein the first device comprises cells of a first type and second device comprises cells of a second type and the library of expression constructs is the same in the first and second devices;
(b) culturing the cells of the first and second devices;
(c) measuring the level of expression of the reporter sequence in each well; and
(d) comparing the level of expression of the reporter sequence to each transcription regulatory sequence between the first cell type and the second cell type. - View Dependent Claims (56, 57)
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64. Analysis software that integrates Z-score transformed promoter activity data with Z-score transformed functional data from DNA methylation experiments, transcription factor binding data, histone modification data, DNase hypersensitivity data, nucleosome displacement data or gene expression data.
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65. A method for determining a methylation pattern in a sequence of nucleic acid comprising:
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(a) creating a first set of labeled nucleic acid segments by;
(i) obtaining a nucleic acid molecule comprising the sequence from a source; and
(ii) labeling the isolated nucleic acid molecule with a first label, whereby labeling creates a first set of labeled nucleic acid segments;
(b) creating a second set of labeled nucleic acid segments by;
(i) obtaining the nucleic acid molecule having the nucleotide sequence from the source;
(ii) contacting the nucleic acid molecule with at least three methyl-sensitive restriction enzymes having different recognition sequences, wherein the enzymes cleave the nucleic acid molecule at un-methylated recognition sequences but not at methylated recognition sequences, thereby nucleic acid fragments;
(iii) isolating nucleic acid fragments of at least 100 nucleotides from the mixture; and
(iv) labeling the fragments with a second, different label, whereby labeling creates a second set of nucleic acid segments;
(c) hybridizing the first and second labeled segments to one or more nucleic acid probes comprising the nucleotide sequence; and
(d) determining areas of the nucleotide sequence that are differentially labeled by the first and second labeled segments, wherein differentially labeled areas are un-methylated areas of the nucleotide sequence. - View Dependent Claims (66, 67, 68, 69, 70)
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Specification