Process for categorizing nucleotide sequence populations
First Claim
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1. A process for categorizing uncharacterized nucleic acid by sorting said nucleic acid into sequence-specific subsets comprising:
- (a) optionally, cleaving said uncharacterized nucleic acid by the action of a reagent, which reagent cleaves said nucleic acid to produce smaller size cleavage products;
(b) reacting either the uncharacterized nucleic acid or said cleavage products from (a) with a first population of adaptor molecules to generate first adaptored products, each of which first adaptor molecules comprises a nucleic acid sequence end recognition means, wherein said population of first adaptor molecules comprises a range of such molecules having sequence end recognition means linking, when present, to a first predetermined subset of said uncharacterized nucleic acid sequences to form first adaptored products;
(c) selecting and separating said first adaptored products resulting from (b);
(d) cleaving adaptors from the selected and separated first adaptored products from (c) to form first selected nucleic acids;
(e) reacting said first selected nucleic acids resulting from (d) with a second population of adaptor molecules to generate second adaptored products, each of which second adaptor molecules comprises nucleic acid end recognition means, wherein said second population of adaptor molecules comprises sequence end recognition means linking to a second predetermined subset of nucleic acid sequences to form second adaptored products; and
(f) selecting and separating said second adaptored products resulting from (e).
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Abstract
A process for the categorization of nucleic acid sequences in which said sequences are linked to a population of adaptor molecules each exhibiting specificity for linking to a sequence including a predetermined nucleotide base, categorization of the resulting linked sequences being based upon selection for the base.
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Citations
41 Claims
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1. A process for categorizing uncharacterized nucleic acid by sorting said nucleic acid into sequence-specific subsets comprising:
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(a) optionally, cleaving said uncharacterized nucleic acid by the action of a reagent, which reagent cleaves said nucleic acid to produce smaller size cleavage products; (b) reacting either the uncharacterized nucleic acid or said cleavage products from (a) with a first population of adaptor molecules to generate first adaptored products, each of which first adaptor molecules comprises a nucleic acid sequence end recognition means, wherein said population of first adaptor molecules comprises a range of such molecules having sequence end recognition means linking, when present, to a first predetermined subset of said uncharacterized nucleic acid sequences to form first adaptored products; (c) selecting and separating said first adaptored products resulting from (b); (d) cleaving adaptors from the selected and separated first adaptored products from (c) to form first selected nucleic acids; (e) reacting said first selected nucleic acids resulting from (d) with a second population of adaptor molecules to generate second adaptored products, each of which second adaptor molecules comprises nucleic acid end recognition means, wherein said second population of adaptor molecules comprises sequence end recognition means linking to a second predetermined subset of nucleic acid sequences to form second adaptored products; and (f) selecting and separating said second adaptored products resulting from (e). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A process of categorizing uncharacterized nucleic acid, comprising:
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(a) cleaving said uncharacterized nucleic acid using an endonuclease which has cleavage and recognition site separated to cleave said nucleic acid to produce double stranded cleavage products, the individual strands of which overlap at cleaved ends to leave a single strand extending to a known extent; b) ligating the cleavage products from (a) with first adaptor molecules to generate first adaptoted cleavage products, each of which first adaptor molecules has a cleavage product end recognition sequence wherein the thus-used first adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a first predetermined subset of the sequences of the cleavage generated extending single strands resulting from (a); (c) selecting and separating the first adaptored cleavage products from (b) which carry an adaptor of first known recognition sequence; (d) cleaving first adaptor molecules from the selected and separated products from (c) to form first selected nucleic acids; (e) ligating the first selected nucleic acids from (d) with second adaptor molecules to generate second adaptoted cleavage products, each of which second adaptor molecules has a cleavage product end recognition sequence wherein the thus-used second adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a second predetermined subset of the sequences of the cleavage-generated extending single strands resulting from (d); and (f) selecting and separating the second adaptoted cleavage products from (e) which comprise an adaptor of second known recognition sequence. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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- 30. A categorizing nucleotide sequence population kit, comprising a population of adaptor molecules in the form of double stranded oligonucleotides having a single stranded end extension as a nucleic acid sequence end recognition means and primers for use in a nucleic acid amplification process, optionally wherein at least some of the adaptor molecules includes a sequence for primer hybridization thereto, wherein the single stranded end extension of said adaptor molecules have recognition means complementary to all possible members of a chosen subset of nucleic acids to be categorized.
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34. A kit of 30, and also including a nucleic acid cleavage reagent.
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35. A process for the categorization of nucleic acid sequences in which said sequences are linked to a population of adaptor molecules each exhibiting specificity for linking to a sequence including a single predetermined nucleotide base, categorization of the resulting linked sequences being based upon selection for the single predetermined nucleotide base.
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36. A process of categorizing uncharacterized nucleic acid comprising:
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(a) cleaving said uncharacterized nucleic acid of an endonuclease having cleavage and recognition site separated, to produce double stranded cleavage products having individual strands of which overlap at cleaved ends to leave a single strand extending to a known length; (b) ligating the cleavage products from (a) with adaptor molecules to generate adaptored cleavage products, each of which adaptor molecules has a cleavage product end recognition sequence, wherein the thus-used adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a predetermined subset of the sequences of the cleavage-generated extending single strands; and (c) selecting and separating adaptored cleavage products resulting from (b) which comprise an adaptor of known recognition sequence wherein said selecting is based upon a chosen base or bases in said recognition sequence which is/are other than the entirety of said recognition sequence.
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37. A process for categorizing uncharacterized nucleic acid by sorting said nucleic acid into sequence-specific subsets comprising:
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(a) optionally, initially cleaving said uncharacterized nucleic acid with a reagent, which cleaves said nucleic acid to produce smaller size cleavage products; (b) reacting either the uncharacterized nucleic acid or cleavage products from (a) with a population of adaptor molecules to generate adaptored products, each of which adaptor molecules comprises a nucleic acid sequence end recognition means, wherein said population of adaptor molecules comprise a range of such molecules having sequence end recognition means linking, when present, to a predetermined subset of nucleic acid sequences; and (c) selecting and separating adaptored products resulting from (b) which include an adaptor of chosen nucleic acid sequence end recognition means, wherein said selecting is based upon a chosen base or bases in the nucleic acid sequence which is recognized by said nucleic acid end recognition means, which base or bases is/are other than the entirety of said recognized nucleic acid sequence.
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38. A method of categorizing or sorting a mixture of uncharacterized nucleic acids into a predetermined subset of nucleic acid sequences, comprising:
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linking, when present, a predetermined subset of nucleic acid sequences of said uncharacterized nucleic acids to a population of adaptor molecules, wherein each adaptor molecule contains a nucleic acid sequence end recognition means recognizing a single and same predetermined base, to form an adaptored product, thereby permitting categorization of said mixture of uncharacterized nucleic acid sequences on the basis of selecting for a subset in which said same and single predetermined base has been recognized, and selecting and separating said adaptored product. - View Dependent Claims (39, 40)
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Specification