Gene expression analysis
First Claim
1. A method of analyzing gene expression in a cell or tissue, the method comprising the steps of(a) forming a population of cDNA molecules from mRNA of a cell or tissue;
- (b) digesting the population of cDNA molecules with at least one restriction endonuclease to produce a population of polynucleotides having predetermined ends;
(c) enzymatically removing a segment of nucleotides from each predetermined end of each polynucleotide and ligating the segments from each end together to form a pair of sequence stages for each polynucleotide, wherein said segments are formed by inserting each of said polynucleotides into a cloning site of a vector, the cloning site being flanked by a first type IIs restriction site and a second type IIs restriction site such that a type IIs restriction endonuclease recognizing either said first or second sites cleaves the vector within to said polynucleotide, the first IIs restriction site and the second type IIs restriction site being the same or different and each of the first and second type IIs restriction sites being unique to the vector;
(d) determining the nucleotide sequences of a sample of pairs of sequence tags; and
(e) tabulating the nucleotide sequences of the pairs of sequence tags to form a frequency distribution of gene expression in the cell or tissue.
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Accused Products
Abstract
The invention provides a method and materials for analyzing the frequency of sequences in a population of polynucleotides, such as a cDNA library. A population of restriction fragments is formed which is inserted into vectors which allow segments to be removed from each end of the inserted fragments. The segments from each restriction fragment are ligated together to form a pair of segments which serves as a tag for the restriction fragment, and the polynucleotide from which the fragment is derived. Pairs of segments are excised from the vectors and ligated to form concatemers which are cloned and sequenced. A tabulation of the sequences of pairs provides a frequency distribution of sequences in the population.
113 Citations
8 Claims
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1. A method of analyzing gene expression in a cell or tissue, the method comprising the steps of
(a) forming a population of cDNA molecules from mRNA of a cell or tissue; -
(b) digesting the population of cDNA molecules with at least one restriction endonuclease to produce a population of polynucleotides having predetermined ends; (c) enzymatically removing a segment of nucleotides from each predetermined end of each polynucleotide and ligating the segments from each end together to form a pair of sequence stages for each polynucleotide, wherein said segments are formed by inserting each of said polynucleotides into a cloning site of a vector, the cloning site being flanked by a first type IIs restriction site and a second type IIs restriction site such that a type IIs restriction endonuclease recognizing either said first or second sites cleaves the vector within to said polynucleotide, the first IIs restriction site and the second type IIs restriction site being the same or different and each of the first and second type IIs restriction sites being unique to the vector; (d) determining the nucleotide sequences of a sample of pairs of sequence tags; and (e) tabulating the nucleotide sequences of the pairs of sequence tags to form a frequency distribution of gene expression in the cell or tissue. - View Dependent Claims (2, 3, 4, 5)
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6. A method of determining sequence frequencies in a population of polynucleotides, the method comprising the steps of:
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(a) providing a population of polynucleotides having predetermined ends; (b) inserting each polynucleotide of the population into a vector, the vector having at least one type IIs restriction endonuclease recognition site adjacent to each end of the inserted polynucleotide, each type IIs restriction endonuclease recognition site being oriented such that a type IIs restriction endonuclease recognizing said sites cleaves the vector within to the inserted polynucleotide; (c) cleaving each vector with one or more type IIs restriction endonucleases recognizing the type IIs restriction endonuclease recognition sites so that the vector is linearized and has a sequence tag of the inserted polynucleotide at each end; (d) re-circularizing the vector to form a pair of sequence tags for the inserted polynucleotide; and (e) determining the nucleotide sequence of each pair of sequence tags of a sample of re-circularized vectors to give the sequence frequencies of the population of polynucleotides. - View Dependent Claims (7, 8)
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Specification