RNA SEQUENCING AND ANALYSIS USING SOLID SUPPORT
First Claim
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1. A method for simultaneous identification and analysis of RNA molecules in a sample, comprising the steps of:
- preparing a solid support which has capturing oligonucleotide molecules attached onto its surface,introducing a functional group at the 3′
end of RNA molecules present in the sample,binding the RNA molecules by hybridization to capturing oligonucleotide molecules which have a sequence complementary to the sequences of the functional group and which are fixed on a solid support, andsubjecting the RNA molecules to analysis as attached to the solid support.
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Abstract
The present invention provides methods for the sequencing of all RNA species within an RNA sample, such as the RNA content obtained from a cell, a tissue, a living organism, or from an artificial source. RNA molecules within the samples are labeled in a RNA-specific manner prior to immobilization on a solid support. One label is used to mark the location of the RNA molecule on the solid support, whereas the second label is used to mark selectively the S′ end of full-length mRNA molecules. RNA molecules are sequenced while being bound to the solid support in one or more sequencing reactions, and sequences of individual RNA molecules can be forwarded to computational analysis for assembling sequence information from individual sequencing reads obtained from the same location on the solid support. Not only unsupervised expression profiling on a genome-wide scale, but also the direct analysis of RNA-RNA interactions become possible as revealed by the analysis of the sequencing information obtained along with genomic information.
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14 Claims
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1. A method for simultaneous identification and analysis of RNA molecules in a sample, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, introducing a functional group at the 3′
end of RNA molecules present in the sample,binding the RNA molecules by hybridization to capturing oligonucleotide molecules which have a sequence complementary to the sequences of the functional group and which are fixed on a solid support, and subjecting the RNA molecules to analysis as attached to the solid support. - View Dependent Claims (2, 3)
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4. A method for simultaneous identification and analysis of RNA molecules in a sample, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, introducing a functional group at the 3′
end of RNA molecules present in the sample,labeling one or more diol groups present in the RNA molecules with a labeling molecule, binding the RNA molecules by hybridization to the capturing oligonucleotide molecules which have a sequence complementary to the sequences of the functional group, detecting the labeling molecule introduced to the RNA molecules to identify a feature, including existence or absence of a Cap structure, of each RNA molecule attached to the solid support, and subjecting the RNA molecules to analysis as attached to the solid support. - View Dependent Claims (5)
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6. A method for simultaneous identification and analysis of mRNA molecules in a sample, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, labeling one or more diol groups present in the mRNA molecules with a labeling molecule, binding the mRNA molecules to the capturing oligonucleotide molecules which have a sequence complementary to a sequence of the 3′
end of the RNA molecules,detecting the labeling molecules attached to the mRNA molecules to identify a feature, including existence or absence of a Cap structure, of each mRNA molecule attached to the solid support, and subjecting the mRNA molecules to analysis as attached to the solid support.
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7. A method for simultaneous identification and analysis of mRNA molecules in a sample, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, labeling the mRNA molecules with a suitable labeling molecule, binding the mRNA molecules by hybridization to the capturing oligonucleotide molecules, which have a sequence complementary to the sequence of the functional group introduced at the 3′
end of the RNA molecules,detecting two labeling molecules for a single mRNA molecule so as to determine that said single RNA molecule is full length having a labeled Cap structure and a labeled polyA tail, and subjecting the full-length mRNA to analysis as attached to the solid support.
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8. A method for simultaneous identification and analysis of mRNA molecules in a sample, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, labeling one or more diol groups present in the mRNA molecules with a suitable labeling molecule, binding the mRNA molecules by hybridization to the capturing oligonucleotide molecules, which have a sequence complementary to the sequence of the functional group introduced at the 3′
end of the RNA molecules,priming reverse transcription of the mRNA molecules attached to the solid support to obtain cDNA strands complementary to the mRNA molecules so as to form DNA-RNA hybrids, subjecting the DNA-RNA hybrids to RNase I treatment for removal of their single stranded portion, detecting the labeling molecule to identify any cDNA strands in the hybrids that have reached the 5′
end of any full-length mRNA,washing away the RNA molecules so as to obtain single-stranded cDNA, and adding a priming site to the single-stranded cDNA molecules which represent full-length mRNA to analysis for sequencing of the 3′
end of such cDNA while it remains attached to the solid support.
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9. A method for simultaneous identification and analysis of RNA molecules in a sample obtained from a single cell or tissue, comprising the steps of:
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preparing a solid support which has capturing oligo-dT oligonucleotide molecules attached onto its surface, modifying RNA molecules in the sample at their 3′
end so as to introduce a functional group in a polyadenylation reaction,extending the RNA molecules having a polyA tail at the 3′
end by adding a polyC tail to their 3′
end,labeling diol groups of RNA molecules within the sample by introducing a fluorescent dye, binding the labeled RNA molecules to a solid support by binding of the functional group to the capturing oligo-dT oligonucleotide molecules on the solid support, detecting labeled RNA molecules on the solid support to determine loading of the solid support and a percentage of capped mRNA molecules within the sample, having the capturing oligonucleotide priming a reverse transcription reaction to synthesize cDNA strands attached to the solid support, removing, by RNase I digestion, labels from 3′
ends of RNA molecules and from Cap structures of DNA-mRNA hybrids that have not extended to reach the 5′
end of mRNA templates,detecting full-length cDNAs for capped mRNAs on the solid support to determine a percentage of capped mRNA or full-length cDNA molecules in the sample, removing the RNA templates, ligating a double-stranded adapter with a priming site to the 3′
end of the single-stranded cDNA attached to the solid support, anddriving, using the priming site within the adapter, a sequence reaction to obtain sequence information from the 5′
end of RNA molecules in the sample as the cDNA molecules remain attached to the solid support.
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10. A method for simultaneous identification and analysis of RNA molecules in a sample obtained from a single cell or tissue, comprising the steps of:
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preparing a solid support which has capturing oligo-dT oligonucleotide molecules attached onto its surface, modifying RNA molecules in the sample at their 3′
end so as to introduce a functional group in a polyadenylation reaction,optionally extending the RNA molecules having a polyA tail at the 3′
end by adding a polyC tail to their 3′
end,labeling diol groups of the RNA molecules by introducing a fluorescent dye, binding the labeled RNA molecules to a solid support by binding of the functional group to the capturing oligo-dT oligonucleotide molecules on the solid support detecting labeled RNA molecules on the solid support to determine loading of the solid support and a percentage of capped mRNA molecules in the sample, having the capturing oligonucleotide prime a reverse transcription reaction to synthesize cDNA strands attached to the solid support and having a sequence complementary to the polyA tail of the RNA template bound to the capturing oligonucleotide, and having the extended capturing oligonucleotide prime a reverse transcription reaction to obtain sequence information from the 3′
end of RNA molecules in the sample as the cDNA strands remain attached to the solid support.
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11. A method for simultaneous identification and analysis of RNA molecules in a sample obtained from a single cell or tissue, comprising the steps of:
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preparing a solid support which has capturing oligo-dT oligonucleotide molecules attached onto its surface, modifying RNA molecules in the sample at their 3′
end so as to introduce a functional group in a polyadenylation reaction,extending RNA molecules having a polyA tail at the 3′
end by adding a polyC tail to their 3′
end.labeling diol groups of the RNA molecules by introducing a fluorescent dye. binding the labeled RNA molecules to a solid support by binding of the functional group to the capturing oligo-dT oligonucleotide molecules on the solid support, detecting labeled RNA molecules on the solid support to determine loading of the solid support and a percentage of capped mRNAs within the sample, having the capturing oligonucleotide prime a reverse transcription reaction to synthesize cDNA molecules attached to the solid support and having a sequence complementary to the polyA tail within the RNA template bound to the capturing oligonucleotide. having the extended capturing oligonucleotide prime a reverse transcription reaction to obtain sequence information from the 3′
end of RNA molecules within the sample.having the extended capturing oligonucleotide prime a reverse transcription reaction to synthesize cDNA strands attached to the solid support and having a sequence complementary to the RNA template bound to the capturing oligonucleotide, removing, by RNase I digestion, labels from 3′
ends of RNA molecules and from Cap structures of cDNA-mRNA hybrids that have not extended to reach the 5′
end of mRNA templates,detecting full-length cDNAs for capped mRNAs on the solid support to determine a percentage of capped mRNAs or full-length cDNAs within the sample. removing RNA templates, ligating a double-stranded adapter with a priming site to the 3′
end of the single-stranded cDNA strand attached to the solid support, anddriving, using the priming site within the adapter, a sequence reaction to obtain sequence information from the 5′
end of RNA molecules in the sample while the cDNA strands remain attached to the solid support.
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12. A method for simultaneous identification and analysis of RNA molecules in a sample obtained from a single cell or tissue, comprising the steps of:
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preparing a solid support which has capturing oligonucleotide molecules attached onto its surface, incubating total RNA molecules contained in the sample with the solid support, having polyadenylated mRNA molecules within the sample hybridize to the capturing oligonucleotide molecules on the solid support while nonpolyadenylated RNA molecules do not bind, washing away RNA molecules that do not bind to the solid support, labeling diol groups in mRNA molecules on the solid support by introducing a fluorescent dye, detecting the labeled mRNA molecules on the solid support to determine loading of the solid support and a percentage of capped mRNAs in the sample, having the capturing oligonucleotide prime a reverse transcription reaction to synthesize cDNA strands attached to the solid support and having a sequence complementary to the polyA tail of the mRNA templates attached to the solid support, having the extended capturing oligonucleotide prime a reverse transcription reaction to obtain sequence information from the 3′
end of mRNA molecules attached to the solid support,having the extended capturing oligonucleotide prime a reverse transcription reaction to synthesize cDNA strands attached to the solid support and having a sequence complementary to the mRNA templates attached to the solid support, removing, by RNase I digestion, labels from 3′
ends of mRNA molecules and from Cap structures of cDNA-mRNA hybrids that have not extended to reach the 5′
end of the mRNA templates,detecting full-length cDNAs for capped mRNAs on the solid support to determine a percentage of capped mRNAs or full-length cDNAs in the sample, removing the RNA templates, ligating a double-stranded adapter with a priming site to the 3′
end of the single-stranded cDNA attached to the solid support, anddriving, using the priming site within the adapter, a sequence reaction to obtain sequence information from the 5′
end of mRNA molecules in the sample while the cDNA strands remain attached to the solid support.
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13. A method for obtaining sequence information from a DNA molecule in a sample in a cyclic reaction comprising the steps of:
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adding to the sample an adapter having a priming site to a 3′
end of a DNA molecule and a recognition site for a restriction endonuclease that cuts outside of its binding site,hybridizing a sequencing primer to the primer site to drive a sequencing reaction, obtaining sequence information from the 3′
end of the DNA molecule,removing a DNA fragment from the 3′
end of the DNA molecule by digestion with the restriction endonuclease that cuts outside of its binding site,adding a new adapter having a priming site and a recognition site for a restriction endonuclease that cuts outside of its binding site to the end of the DNA molecule opened by the removing step, and repeating the above steps.
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14. A method for detecting RNA molecules having one or more diol groups, comprising the steps of:
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introducing a labeling molecule to one or more diol groups of an RNA molecule so as to obtain labeled RNA, adding the labeled RNA to a sample, and detecting the labeling molecule for identifying the RNA molecule in the sample.
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Specification
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Current AssigneeKabushiki Kaisha Dnaform, Riken
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Original AssigneeKabushiki Kaisha Dnaform, Riken
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InventorsCarninci, Piero, Katayama, Mutsumi, Katayama, Shintaro, Hayashizaki, Yoshihide, Harbers, Matthias, Itoh, Masayoshi
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Application NumberUS12/186,009Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/6809 Methods for determination o...C12Q 1/6834 Enzymatic or biochemical co...C12Q 1/6874 involving nucleic acid arra...C12Q 2525/155 incorporating/generating a ...C12Q 2535/101 Sanger sequencing method, i...C12Q 2565/537 characterised by the captur...
