Nucleic acid indexing
First Claim
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1. A method for indexing polynucleotides, comprising the steps of:
- (A) combining under base-pairing conditions;
(i) one or more distinguishable sets of indexing adaptors, each adaptor comprising a sequence for amplification and at least one terminus having a single-stranded region characterized by, in non-overlapping order inward from the end;
(a) an indexing sequence n bases long and (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer and wherein further each set of adaptors comprises one or more indexing sequences, and (ii) one or more corresponding polynucleotides, each of which comprises at least one terminus characterized by, in non-overlapping order inward from the end;
(a) a region having a sequence characteristic of cleavage by a Class II restriction endonuclease and (b) a double-stranded region having on one strand a sequence that base-pairs with an indexing sequence of an adaptor and on the other strand a sequence complementary thereto;
(B) base-pairing at least one adaptor terminus and at least one corresponding polynucleotide terminus to form at least one strand-displaced structure wherein the indexing sequence of the single-stranded region of the adaptor terminus is base-paired with the sequence that base-pairs with an indexing sequence of the terminus of the corresponding polynucleotide, and the complementary sequence on the other strand of the polynucleotide terminus is displaced from base-pairing thereto;
(C) for each adaptor set, amplifying the corresponding polynucleotides that form strand-displaced structures using the sequence for amplification; and
(D) distinguishing the amplified corresponding polynucleotides thereby indexing the polynucleotides by their base-pairing to the distinguishable sets of adaptors.
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Abstract
A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known. Profiling methods and other methods for characterizing polynucleotides are presented.
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Citations
29 Claims
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1. A method for indexing polynucleotides, comprising the steps of:
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(A) combining under base-pairing conditions;
(i) one or more distinguishable sets of indexing adaptors, each adaptor comprising a sequence for amplification and at least one terminus having a single-stranded region characterized by, in non-overlapping order inward from the end;
(a) an indexing sequence n bases long and (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer and wherein further each set of adaptors comprises one or more indexing sequences, and(ii) one or more corresponding polynucleotides, each of which comprises at least one terminus characterized by, in non-overlapping order inward from the end;
(a) a region having a sequence characteristic of cleavage by a Class II restriction endonuclease and (b) a double-stranded region having on one strand a sequence that base-pairs with an indexing sequence of an adaptor and on the other strand a sequence complementary thereto;
(B) base-pairing at least one adaptor terminus and at least one corresponding polynucleotide terminus to form at least one strand-displaced structure wherein the indexing sequence of the single-stranded region of the adaptor terminus is base-paired with the sequence that base-pairs with an indexing sequence of the terminus of the corresponding polynucleotide, and the complementary sequence on the other strand of the polynucleotide terminus is displaced from base-pairing thereto;
(C) for each adaptor set, amplifying the corresponding polynucleotides that form strand-displaced structures using the sequence for amplification; and
(D) distinguishing the amplified corresponding polynucleotides thereby indexing the polynucleotides by their base-pairing to the distinguishable sets of adaptors. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for characterizing a population of polynucleotides, comprising the steps of:
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(A) combining under base-pairing conditions;
(i) one or more distinguishable sets of indexing adaptors, each adaptor comprising a sequence for amplification and at least one terminus having a single-stranded region characterized by, in non-overlapping order inward from the end;
(a) an indexing sequence n bases long and (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer and wherein further each set of adaptors comprises one or more indexing sequences, and(ii) a population of polynucleotides that includes one or more corresponding polynucleotides, each of which comprises at least one terminus characterized by, in non-overlapping order inward from the end;
(a) a region having a sequence characteristic of cleavage by a Class II restriction endonuclease and (b) a double-stranded region having on one strand a sequence that base-pairs with an indexing sequence of an adaptor and on the other strand a sequence complementary thereto;
(B) base-pairing at least one adaptor terminus to at least one corresponding polynucleotide terminus to form at least one strand-displaced structure wherein the indexing sequence of the single-stranded region of the adaptor terminus is base-paired with the sequence that base-pairs with an indexing sequence of the terminus of the corresponding polynucleotide, and the complementary sequence on the other strand of the polynucleotide terminus is displaced from base-pairing thereto;
(C) for each adaptor set, amplifying corresponding polynucleotides that form strand-displaced structures using the sequence for amplification;
(D) determining one or more amplified corresponding polynucleotides or the absence thereof; and
(E) characterizing the population of polynucleotides by the amplified corresponding polynucleotides that form strand-displaced structures using the sequence for amplification, or the absence thereof. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification