Method of constructing cdna tag for identifying expressed gene and method of analyzing gene expression
First Claim
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1. A method for the preparation of cDNA tags for identifying expressed genes comprising:
- providing complementary deoxyribonucleic acids (cDNAs);
cleaving the cDNAs with a type II restriction enzyme to prepare cDNA fragments;
ligating the cDNA fragments to linker Xes which have a recognition site of a first type IIS restriction enzyme and which form a recognition site of a second type IIS restriction enzyme at the site linking with the cleavage end-sites of the cDNA fragments formed by the type II restriction enzyme to prepare linker X-cDNA fragment complexes;
cleaving the linker X-cDNA fragment complexes with the second type II restriction enzyme to prepare linker X-cDNA tag complexes;
ligating linker Ys which have a recognition site of the first type IIS restriction enzyme to the cleavage end-sites of the linker X-cDNA tag complexes formed by the second type IIS restriction enzyme to prepare linker X-cDNA-tag-linker Y complexes;
amplifying the linker X-cDNA fragment-linker Y complexes; and
cleaving the amplified products thus obtained with the first type IIS restriction enzymes simultaneously or in turn to prepare the cDNA tags for identifying expressed genes.
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Abstract
There are provided a method for the preparation of cDNA tags for identifying expressed genes and a method for analysis of gene expression. The cDNA tags for identifying expressed genes are prepared by the method comprising a kind of type II restriction enzyme, two kinds of type IIS restriction enzymes and linkers X and Y having a recognition site for one of two kinds of type IIS restriction enzymes. The cDNA tags can be used alone or in combination like chain (concatemer) formed by combining process to analyze gene expression.
6 Citations
50 Claims
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1. A method for the preparation of cDNA tags for identifying expressed genes comprising:
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providing complementary deoxyribonucleic acids (cDNAs);
cleaving the cDNAs with a type II restriction enzyme to prepare cDNA fragments;
ligating the cDNA fragments to linker Xes which have a recognition site of a first type IIS restriction enzyme and which form a recognition site of a second type IIS restriction enzyme at the site linking with the cleavage end-sites of the cDNA fragments formed by the type II restriction enzyme to prepare linker X-cDNA fragment complexes;
cleaving the linker X-cDNA fragment complexes with the second type II restriction enzyme to prepare linker X-cDNA tag complexes;
ligating linker Ys which have a recognition site of the first type IIS restriction enzyme to the cleavage end-sites of the linker X-cDNA tag complexes formed by the second type IIS restriction enzyme to prepare linker X-cDNA-tag-linker Y complexes;
amplifying the linker X-cDNA fragment-linker Y complexes; and
cleaving the amplified products thus obtained with the first type IIS restriction enzymes simultaneously or in turn to prepare the cDNA tags for identifying expressed genes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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- 27. Linker X comprising a recognition site of the first type IIS restriction enzymes and being capable of forming a recognition site of the second type IIS restriction enzyme at the position linking to the cDNA fragment cleaved by the type II restriction enzyme.
- 29. Linker X-cDNA fragment complex comprising cDNA fragment formed by cleaving with a type II restriction enzyme and linker X having a recognition site of the first type IIS restriction enzymes and being capable of forming a recognition site of the second type IIS restriction enzyme at the position linking to the cDNA fragment cleaved by the type II restriction enzyme.
- 49. A kit for the preparation of cDNA tags for identifying expressed genes wherein the kit comprises a type II restriction enzyme, a first type IIS restriction enzyme, a second type IIS restriction enzyme, linker Xes which have a recognition site of the first type IIS restriction enzyme and which form a recognition site of the second type IIS restriction enzyme at the site linking with the cleavage end of the cDNA fragments formed by the type II restriction enzyme, and linker Ys which have a recognition site of the first type IIS restriction enzyme.
Specification