Method of utilizing the 5'end of transcribed nucleic acid regions for cloning and analysis
First Claim
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1. A method for preparing a DNA fragment corresponding to a nucleotide sequence of a 5′
- end region of an mRNA, comprising the steps of;
(a) preparing a nucleic acid corresponding to a nucleotide sequence of the 5′
end of an mRNA;
(b) attaching at least one linker to the nucleic acid;
(c) cleaving the nucleic acid with a restriction enzyme having its recognition site within the linker and its cleavage site within the nucleic acid corresponding to the 5′
end of the mRNA; and
(d) collecting a resulting DNA fragment corresponding to the 5′
end of the mRNA.
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Abstract
A method is disclosed for obtaining the 5′ends of transcribed regions from a plurality of nucleic acid fragments obtained from biological materials or synthetic pools. DNA fragments encoding the 5′ends are enriched for their individual analysis or for the analysis of concatemers thereof. The sequence information derived from 5′ ends can be used for characterization and cloning of the transcriptome.
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58 Claims
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1. A method for preparing a DNA fragment corresponding to a nucleotide sequence of a 5′
- end region of an mRNA, comprising the steps of;
(a) preparing a nucleic acid corresponding to a nucleotide sequence of the 5′
end of an mRNA;
(b) attaching at least one linker to the nucleic acid;
(c) cleaving the nucleic acid with a restriction enzyme having its recognition site within the linker and its cleavage site within the nucleic acid corresponding to the 5′
end of the mRNA; and
(d) collecting a resulting DNA fragment corresponding to the 5′
end of the mRNA. - View Dependent Claims (2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58)
- end region of an mRNA, comprising the steps of;
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7. A method for preparing a DNA fragment corresponding to a nucleotide sequence of a 5′
- end region of an mRNA, comprising steps of;
(a) substituting a cap structure of an mRNA with an oligonucleotide, wherein the oligonucleotide comprises a restriction enzyme recognition site, and a cleavage site of a restriction enzyme is within the nucleic acid corresponding to the 5′
end of the mRNA;
(b) synthesizing a first strand cDNA using the mRNA as a template;
(c) synthesizing a second strand cDNA using the first stand cDNA as a template;
(d) cleaving a resulting double stranded cDNA with the restriction enzyme; and
(e) collecting a resulting DNA fragment corresponding to 5′
end of the mRNA. - View Dependent Claims (56)
- end region of an mRNA, comprising steps of;
Specification