Method for serial analysis of gene expression
First Claim
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1. An isolated oligonucleotide composition derived from cDNA, said composition comprising:
- at least two different defined nucleotide sequence tags, wherein each defined nucleotide sequence tag consists of about 6 to 30 nucleotides of said cDNA 5'"'"' of the 5'"'"'-most cleavage site of a restriction endonuclease within said cDNA or 3'"'"' of the 3'"'"'-most cleavage site of a restriction endonuclease within said cDNA;
wherein at least one tag corresponds to at least one expressed gene.
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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
44 Claims
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1. An isolated oligonucleotide composition derived from cDNA, said composition comprising:
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at least two different defined nucleotide sequence tags, wherein each defined nucleotide sequence tag consists of about 6 to 30 nucleotides of said cDNA 5'"'"' of the 5'"'"'-most cleavage site of a restriction endonuclease within said cDNA or 3'"'"' of the 3'"'"'-most cleavage site of a restriction endonuclease within said cDNA; wherein at least one tag corresponds to at least one expressed gene. - View Dependent Claims (2, 3)
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4. A method for the detection of gene expression comprising:
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providing complementary deoxyribonucleic acid (cDNA) oligonucleotides; cleaving said cDNA oligonucleotides with a restriction enzyme at a first restriction endonuclease site to provide cDNA fragments; isolating the 5'"'"' or 3'"'"' end of said cDNA fragments to provide defined nucleotide sequence tags to said oligonucleotides of said fragments; isolating a first define nucleotides sequence tag from a first cDNA oligonucleotides and second defined nucleotide sequence tag from a second cDNA oligonucleotides; linking the first tag to a first oligonucleotides linker, wherein the first oligonucleotides linker comprises a first sequence for hybridization of an amplification primer and linking the second tag to a second tag to a second oligonucleotides linker, wherein the second oligonucleotides linker comprises a second sequence for hybridization of an amplification primer; and determining the nucleotide sequence of a tag, wherein said tag corresponds to an expressed gene. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. 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 at the 5'"'"' or 3'"'"' terminus of the cDNA thereby producing defined sequence tags; isolating the defined sequence tags; dividing said defined sequence tags into first and second pools; ligating a first pool of tags with a first oligonucleotide linker having a first sequence capable of hybridizing to an amplification primer and ligating a second pool of tags with a second oligonucleotide linker having a second sequence capable of hybridizing to an amplification primer; cleaving the tags with a second restriction endonuclease which cleaves at a position outside its recognition sequence; ligating the two pools of tags to produce a ditag; and determining the nucleotide sequence of the tag(s), wherein the tag(s) correspond to a mRNA from an expressed gene. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39)
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34. The method of claim 34, wherein the ditag is about 14 to 22 base pairs.
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40. 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 a first container containing a first oligonucleotide linker having a first hybridization sequence which hybridizes an amplification primer;
- a second container containing a second oligonucleotide linkers having a second hybridization sequence which hybridizes to an amplification primer, wherein the linkers further comprise a restriction endonuclease recognition site; and
third and fourth containers having nucleic acid primers for hybridization to the first and second hybridization sequences of the linkers. - View Dependent Claims (41, 42, 43, 44)
- a second container containing a second oligonucleotide linkers having a second hybridization sequence which hybridizes to an amplification primer, wherein the linkers further comprise a restriction endonuclease recognition site; and
Specification