Method of identifying gene transcription patterns
First Claim
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1. A method of obtaining a set of tags from a source of interest comprising the steps of:
- (a) providing a double-stranded cDNA, having a poly A or a poly T tail, (b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment, (c) ligating to said cDNA fragment a 5′
adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct, (d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct, (e) ligating to said second cDNA construct a 3′
adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3′
adjacent to said second cDNA construct, providing a third cDNA construct, (f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and (g) determining the nucleotide sequences of said set of tags.
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Abstract
This invention provides an rapid, artifact free, improved method of obtaining short DNA “tag” or arrays thereof, allowing for determination of the relative abundance of a gene transcript within a given mRNA population and is useful to identify patterns of gene transcription, as well as identify new genes.
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Citations
8 Claims
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1. A method of obtaining a set of tags from a source of interest comprising the steps of:
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(a) providing a double-stranded cDNA, having a poly A or a poly T tail, (b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment, (c) ligating to said cDNA fragment a 5′
adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct,(d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct, (e) ligating to said second cDNA construct a 3′
adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3′
adjacent to said second cDNA construct, providing a third cDNA construct,(f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and (g) determining the nucleotide sequences of said set of tags. - View Dependent Claims (2, 3, 4)
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5. A method of obtaining an array of tags, comprising the steps of:
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(a) providing double-stranded cDNA from an mRNA using a biotinylated oligo dT primer, (b) cleaving within said double-stranded cDNA with a punctuating restriction endonuclease providing a population of a cDNA fragments, (c) ligating to said cDNA fragment a 5′
adapter comprising,i) a single-stranded end compatible with ends produced by cleavage with said punctuating restriction endonuclease;
ii) a recognition sequence for a type IIs restriction endonuclease located 5′
to said single-stranded end;
iii) a recognition sequence for a 5′
-cloning restriction endonuclease located 5′
to a recognition sequence for said type IIs restriction endonuclease, providing a first cDNA construct,(d) isolating said first cDNA construct by affinity capture on solid phase streptavidin, (e) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct, (f) ligating to said second cDNA construct a 3′
adapter comprising,i) a degenerate single-stranded end compatible with ends produced by said type IIs restriction endonuclease;
ii) a recognition sequence for said punctuating restriction endonuclease located 3′
to said degenerate end;
iii) a recognition sequence for a 3′
-cloning restriction endonuclease located 3′
to a recognition sequence for said punctuating restriction endonuclease, providing a third cDNA construct,(g) digesting said third cDNA construct with said 5′ and
3′
-cloning restriction endonucleases providing a fourth cDNA construct;
(h) inserting said fourth cDNA construct into a cloning vector digested with said 5′ and
3′
-cloning restriction endonucleases,(i) replicating said vector DNA in a suitable host strain, (j) isolating said vector DNA, (k) digesting said vector DNA with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences and a GC clamp, (l) ligating said set of tags providing arrays of tags comprising at least 10 tags and GC rich clamps, (m) optionally inserting said arrays of tags into a sequence vector, and (n) optionally determining the nucleotide sequences of the arrays of tags. - View Dependent Claims (6, 7)
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8. A method of obtaining a set of tags from a source of interest comprising the steps of:
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(a) providing a double-stranded cDNA, prepared from mRNA, (b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment, (c) ligating to said cDNA fragment a 5′
adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct,(d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct, (e) ligating to said second cDNA construct a 3′
adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3′
adjacent to said second cDNA construct, providing a third cDNA construct,(f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and (g) determining the nucleotide sequences of said set of tags.
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Specification