Method for serial analysis of gene expression
First Claim
1. An isolated oligonucleotide composition comprising at least one ditag, wherein the ditag comprises two covalently joined defined nucleotide sequence tags in opposite orientation, wherein each tag corresponds to at least one expressed gene.
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Accused Products
Abstract
Serial analysis of gene expression, SAGE, a method for the rapid quantitative and qualitative analysis of transcripts is provided. Short defined sequence tags corresponding to expressed genes are isolated and analyzed. Sequencing of over 1,000 defined tags in a short period of time (e.g., hours) reveals a gene expression pattern characteristic of the function of a cell or tissue. Moreover, SAGE is useful as a gene discovery tool for the identification and isolation of novel sequence tags corresponding to novel transcripts and genes.
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Citations
43 Claims
- 1. An isolated oligonucleotide composition comprising at least one ditag, wherein the ditag comprises two covalently joined defined nucleotide sequence tags in opposite orientation, wherein each tag corresponds to at least one expressed gene.
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4. A method for the detection of gene expression comprising:
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producing complementary deoxyribonucleic acid (cDNA) oligonucleotides; isolating a first defined nucleotide sequence tag from a first cDNA oligonucleotide and a second defined nucleotide sequence tag from a second cDNA oligonucleotide; linking the first tag to a first oligonucleotide linker thereby forming a first linked nucleic acid, wherein the first oligonucleotide linker comprises a first enzyme recognition site that allows DNA cleavage at a site in the first defined nucleotide sequence distant from the first recognition site; linking the second tag to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second enzyme recognition site that allows DNA cleavage at a site in the second defined nucleotide sequence distant from the second recognition site; cleaving the first and the second linked nucleic acids with at least one enzyme that recognizes each of the recognition sites; ligating the first and second tags to form a ditag; and determining the nucleotide sequence of at least one tag of the ditag to detect gene expression. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method for detection of gene expression comprising:
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cleaving a cDNA sample with a first restriction endonuclease, wherein the endonuclease cleaves the cDNA at a defined position in the cDNA thereby producing defined sequence tags; isolating the defined cDNA tags and forming a first pool of tags; ligating a first pool of tags with a first oligonucleotide linker having a first enzyme recognition site that allows DNA cleavage at a site distant from the second recognition site; ligating a second pool of tags with a second oligonucleotide linker having a second enzyme recognition site that allows DNA cleavage at a site distant from the second recognition site; cleaving the tags with a first and a second tag cleaving restriction endonuclease, wherein the first tag-cleaving restriction endonuclease recognizes a first enzyme recognition site and cleaves at a site distant from the first recognition site and wherein the second tag-cleaving restriction endonuclease recognizes a second enzyme recognition site and cleaves at a site distant from the second recognition site; ligating the two pools of tags to produce at least one ditag; and determining the nucleotide sequence of at least one ditag, wherein the ditag(s) correspond to sequence from an expressed gene. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 43)
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38. A kit useful for detection of gene expression wherein the presence of a cDNA ditag is indicative of expression of a gene having a sequence of a tag of the ditag, the kit comprising one or more containers comprising a first container containing a first oligonucleotide linker having a first sequence useful hybridization of an amplification primer;
- a second container containing a second oligonucleotide linker having a second oligonucleotide linker having a second sequence useful hybridization of an amplification primer, wherein the linkers further comprise a restriction endonuclease site for cleavage of DNA at a site distant from the restriction endonuclease recognition site; and
a third and fourth container having a nucleic acid primers for hybridization to the first and second unique sequences of the linker. - View Dependent Claims (39, 40, 41, 42)
- a second container containing a second oligonucleotide linker having a second oligonucleotide linker having a second sequence useful hybridization of an amplification primer, wherein the linkers further comprise a restriction endonuclease site for cleavage of DNA at a site distant from the restriction endonuclease recognition site; and
Specification