Use of representations of DNA for genetic analysis
First Claim
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1. A method of producing a compound representation of DNA, comprising:
- (a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments;
(b) ligating an adaptor to said digested DNA fragments;
(c) amplifying DNA fragments created in step b) using primers complementary to said adaptor to provide a first representation of said DNA;
(d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; and
(e) amplifying said DNA fragments of step (d) using primers which are the same as the primers used in step c) to provide a compound representation of said DNA.
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Abstract
It is an object of the present invention to provide a solution to problems associated with the use of microarray technology for the analysis DNA. The present invention provides compositions and methods for the use of simple and compound representations of DNA in microarray technology. The present invention is also directed to methods for the production of High Complexity Representations (HCRs) of the DNA from cells.
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Citations
15 Claims
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1. A method of producing a compound representation of DNA, comprising:
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(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments; (b) ligating an adaptor to said digested DNA fragments; (c) amplifying DNA fragments created in step b) using primers complementary to said adaptor to provide a first representation of said DNA; (d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; and (e) amplifying said DNA fragments of step (d) using primers which are the same as the primers used in step c) to provide a compound representation of said DNA. - View Dependent Claims (4, 5, 12)
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2. A method of producing a compound representation of DNA, comprising:
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(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments; (b) ligating a first adaptor to said digested DNA fragments; (c) amplifying DNA fragments created in step b) using primers complementary to said first adaptor to provide a first representation of said DNA; (d) digesting the first representation with a second restriction endonuclease to provide digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; (e) ligating a second adaptor to the ends of said digested DNA fragments created by the second restriction endonuclease in step d); (f) ligating a third adaptor to the 5′
ends of DNA fragments create in step c) and not cleaved by the second restriction endonuclease in step (d); and(g) amplifying said DNA fragments of step (e) and (f) using primers complementary to the second and third adaptors to provide a compound representation of said DNA. - View Dependent Claims (6, 7, 13)
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3. A method of producing a compound representation of DNA, comprising:
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(a) digesting a sample of DNA with a first restriction endonuclease to provide digested DNA fragments; (b) ligating an adaptor to said digested DNA fragments; (c) digesting DNA fragments created in step b) with a second restriction endonuclease to provide double-digested DNA fragments, wherein said second restriction endonuclease is different from said first restriction endonuclease; and (d) amplifying said double-digested DNA fragments using primers complementary to said adaptor to provide a compound representation of said DNA. - View Dependent Claims (8, 9, 14)
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10. A method of producing a high complexity representation of DNA, comprising:
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(a) digesting a sample of DNA with at least two restriction endonucleases to provide digested DNA fragments in which at least about 20% of said fragments are at between 200 and 1200 bp, wherein the second restriction endonuclease is different from the first restriction endonuclease; (b) ligating adaptors to said digested DNA fragments; and (c) amplifying DNA fragments created in step b) using primers complementary to said adaptors to provide a high complexity representation of said DNA. - View Dependent Claims (11, 15)
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Specification