Serial analysis of transcript expression using MmeI and long tags
First Claim
1. In a method for detecting expressed transcripts in which a first defined nucleotide sequence tag is isolated from a first cDNA oligonucleotide and a second defined nucleotide sequence tag is isolated from a second cDNA oligonucleotide, and the first defined nucleotide sequence tag is linked to a first oligonucleotide linker thereby forming a first linked nucleic acid, wherein the first oligonucleotide linker comprises a recognition site for a restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the first recognition site;
- and the second defined nucleotide sequence tag is linked to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for the restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the second recognition site;
wherein the first and the second linked nucleic acids are cleaved with said restriction endonuclease;
wherein the first and second tags are ligated to form ditags; and
the nucleotide sequence of at least one tag of the ditag is determined to detect gene expression, the improvement comprising;
using MmeI as the restriction endonuclease to form 3′
overhanging ends on said first and second tags.
2 Assignments
0 Petitions
Accused Products
Abstract
Serial analysis of gene expression, SAGE, a method for the rapid quantitative and qualitative analysis of transcripts, has been improved to provide more genetic information about each analyzed transcript by use of MmeI restriction endonuclease. In SAGE, 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.
-
Citations
30 Claims
-
1. In a method for detecting expressed transcripts in which a first defined nucleotide sequence tag is isolated from a first cDNA oligonucleotide and a second defined nucleotide sequence tag is isolated from a second cDNA oligonucleotide, and the first defined nucleotide sequence tag is linked to a first oligonucleotide linker thereby forming a first linked nucleic acid, wherein the first oligonucleotide linker comprises a recognition site for a restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the first recognition site;
- and the second defined nucleotide sequence tag is linked to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for the restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the second recognition site;
wherein the first and the second linked nucleic acids are cleaved with said restriction endonuclease;
wherein the first and second tags are ligated to form ditags; and
the nucleotide sequence of at least one tag of the ditag is determined to detect gene expression, the improvement comprising;using MmeI as the restriction endonuclease to form 3′
overhanging ends on said first and second tags.
- and the second defined nucleotide sequence tag is linked to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for the restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the second recognition site;
-
2. A method for the detection of transcript expression comprising:
-
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 recognition site for MmeI restriction endonuclease;
linking the second tag to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for MmeI restriction endonuclease;
cleaving the first and the second linked nucleic acids with MmeI restriction endonculease to form 3′
overhanging ends;
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 transcript expression. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
-
-
14. A method for detection of transcript expression comprising:
-
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 pool of tags;
ligating the pool of tags with oligonucleotide linkers having a recognition site for a second restriction endonuclease which is MmeI which forms 3′
overhanging ends;
cleaving the tags with MmeI restriction endonuclease to form 3′
overhanging ends;
ligating the pool of tags to produce at least one ditag; and
determining the nucleotide sequence of at least one ditag, wherein the nucleotide sequence of the ditag corresponds to sequence from at least one expressed transcripts. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
-
Specification