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US 6,280,948 B1Granted
Nucleic acid indexing
AssigneeWisconsin Alumni Research Foundation
InventorsRichard A. Guilfoyle, Zhen Guo
Life of this patent
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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.
Claims29
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.
6 more elements
A method for characterizing a population of 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) 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.
7 more elements
Dependencies are inferred from the claim text.