Sequencing by proxy
First Claim
1. A method of simultaneously determining a signature sequence for each polynucleotide in a population of polynucleotides, the method comprising the steps of:
- attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially every different polynucleotide has a different oligonucleotide tag attached;
generating a size ladder of polynucleotide fragments for each tag-polynucleotide conjugate, each polynucleotide fragment of the same size ladder having an end and the same oligonucleotide tag as every other polynucleotide fragment of the size ladder;
separating the polynucleotide fragments into size classes;
labeling the oligonucleotide tag of each polynucleotide fragment according to the identity of one or more nucleotides at the end of such polynucleotide fragment;
copying the labeled oligonucleotide tags of each polynucleotide fragment of each size class; and
separately hybridizing the labeled oligonucleotide tags of each size class with their respective complements under stringent hybridizaion conditions, the respective complements being attached as populations of substantially identical oligonucleotides in spatially discrete and addressable regions on one or more solid phase supports, and the respective signature sequences being determined by the sequence of labels associated with each spatially discrete and addressable region of the one or more solid phase supports.
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Accused Products
Abstract
The invention provides methods, kits and materials for determinining simultaneously signature sequences of a population of tagged polynucleotides. Size ladders of polynucleotide fragments are generated from the population of tagged polynucleotides that contain a plurality of size classes. After the size classes are separated, tags of the separated fragment are copied and labeled according to the identity of one or more bases at the ends of the fragments. In a preferred embodiment, the labeled tags are then specifically hybridized to plurality of identical microarrays of tag complements such that the tags from different size classes are hybridized to separate microarrays. Signature sequences are determined by signals generated at hybridization sites having the same address on each of the plurality of microarrays.
59 Citations
16 Claims
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1. A method of simultaneously determining a signature sequence for each polynucleotide in a population of polynucleotides, the method comprising the steps of:
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attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially every different polynucleotide has a different oligonucleotide tag attached;
generating a size ladder of polynucleotide fragments for each tag-polynucleotide conjugate, each polynucleotide fragment of the same size ladder having an end and the same oligonucleotide tag as every other polynucleotide fragment of the size ladder;
separating the polynucleotide fragments into size classes;
labeling the oligonucleotide tag of each polynucleotide fragment according to the identity of one or more nucleotides at the end of such polynucleotide fragment;
copying the labeled oligonucleotide tags of each polynucleotide fragment of each size class; and
separately hybridizing the labeled oligonucleotide tags of each size class with their respective complements under stringent hybridizaion conditions, the respective complements being attached as populations of substantially identical oligonucleotides in spatially discrete and addressable regions on one or more solid phase supports, and the respective signature sequences being determined by the sequence of labels associated with each spatially discrete and addressable region of the one or more solid phase supports. - View Dependent Claims (2, 3, 4, 5)
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6. A method of simultaneously determining a signature sequence of each polynucleotide in a sample of tag-polynucleotide conjugates wherein substantially every different polynucleotide has a different tag, the method comprising the steps of:
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generating a size ladder for every tag-polynucleotide conjugate such that each size ladder has a plurality of size classes of polynucleotide fragments;
separating the size classes of polynucleotide fragments;
amplifying and labeling the tag of each polynucleotide fragment according to the identity of one or more nucleotides at an end of each such polynucleotide fragment;
separately hybridizing the labeled tags of each size class with their respective complements under stringent hybridizaion conditions, the respective complements being attached to each of a plurality of microarrays, each microarray of the plurality having the same spatially addressable hybridization sites; and
determining each signature sequence in the sample by a set of signals generated at hybridization sites having the same address on each of the plurality of microarrays. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14)
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15. A kit for simultaneously determining a signature sequence of each polynucleotide in a sample of tag-polynucleotide conjugates wherein substantially every different polynucleotide has a different tag, the kit comprising:
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a vector containing a repertoire of oligonucleotide tags for forming tag-polynucleotide conjugates;
extension oligonucleotides for extending an initializing oligonucleotide to generate size ladders of polynucleotide fragments; and
a plurality of microarrays of tag complements. - View Dependent Claims (16)
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Specification