Methods for selecting and amplifying polynucleotides
First Claim
1. A method of selecting and amplifying polynucleotides on a solid support, comprising:
- a) providing a plurality of amplification oligonucleotides immobilized on a solid support;
b) hybridizing a population of oligonucleotide probes to a subset of said amplification oligonucleotides, each of said oligonucleotide probes comprising a first portion which is complementary to the amplification oligonucleotides and a second portion which comprises sequence from a selected region of a template polynucleotide;
c) performing an extension reaction to extend hybridised amplification oligonucleotides to produce a population of support-bound capture oligonucleotides and a population of unextended amplification oligonucleotides, each capture oligonucleotide in said population comprising a sequence that is complementary to a selected region of a template polynucleotide;
d) applying a population of template polynucleotides to the solid support under conditions such that the template polynucleotides selectively hybridise to the support-bound capture oligonucleotides;
e) extending the support-bound capture oligonucleotides that are hybridized to the template polynucleotides, thereby generating extension products, the extension products having a portion complementary to the template polynucleotides and a portion complementary to the unextended amplification oligonucleotides, wherein the unextended amplification oligonucleotides are reversibly blocked during the extension of the capture oligonucleotides;
f) amplifying the extension products, wherein the amplifying comprises annealing one or more of the unextended amplification oligonucleotides to one or more of the extension products, thereby producing a solid-phase amplification product; and
g) sequencing a first strand of the solid-phase amplification product of step f) to obtain at least part of the nucleotide sequence of the template polynucleotide.
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Abstract
The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.
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Citations
18 Claims
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1. A method of selecting and amplifying polynucleotides on a solid support, comprising:
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a) providing a plurality of amplification oligonucleotides immobilized on a solid support; b) hybridizing a population of oligonucleotide probes to a subset of said amplification oligonucleotides, each of said oligonucleotide probes comprising a first portion which is complementary to the amplification oligonucleotides and a second portion which comprises sequence from a selected region of a template polynucleotide; c) performing an extension reaction to extend hybridised amplification oligonucleotides to produce a population of support-bound capture oligonucleotides and a population of unextended amplification oligonucleotides, each capture oligonucleotide in said population comprising a sequence that is complementary to a selected region of a template polynucleotide; d) applying a population of template polynucleotides to the solid support under conditions such that the template polynucleotides selectively hybridise to the support-bound capture oligonucleotides; e) extending the support-bound capture oligonucleotides that are hybridized to the template polynucleotides, thereby generating extension products, the extension products having a portion complementary to the template polynucleotides and a portion complementary to the unextended amplification oligonucleotides, wherein the unextended amplification oligonucleotides are reversibly blocked during the extension of the capture oligonucleotides; f) amplifying the extension products, wherein the amplifying comprises annealing one or more of the unextended amplification oligonucleotides to one or more of the extension products, thereby producing a solid-phase amplification product; and g) sequencing a first strand of the solid-phase amplification product of step f) to obtain at least part of the nucleotide sequence of the template polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of capturing and amplifying a selected sequence comprising:
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a) obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides, wherein the members of said first and second populations of surface-bound oligonucleotides are attached to the same substrate but not spatially addressed on said substrate; b) hybridizing a first member of said first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with said first member and a region that contains a genomic sequence, c) extending said first member of said first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to said genomic sequence; d) hybridizing said support-bound selection primer to a nucleic acid fragment comprising said genomic sequence; e) extending said support-bound selection primer to produce an extension product that contains the complement of a sequence that flanks said genomic sequence; and f) annealing said extension product to an unextended member of said first and second populations of surface-bound oligonucleotides and amplifying said extension product on said substrate using unextended members of said first and second populations of surface-bound oligonucleotides, to produce a PCR product. - View Dependent Claims (17)
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18. A method of capturing and amplifying a selected sequence comprising the following steps, in order:
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a) obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides, wherein the members of said first and second populations of surface-bound oligonucleotides are attached to the same substrate but not spatially addressed on said substrate; b) hybridizing a first member of said first population of surface-bound oligonucleotides to a synthetically-made selection oligonucleotide comprising a region that hybridizes with said first member and a region that contains a genomic sequence, c) extending said first member of said first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to said genomic sequence; d) denaturing the selection oligonucleotide from the support-bound selection primer; e) hybridizing said support-bound selection primer to a fragmented genome to produce a duplex comprising said support-bound selection primer and a fragment of genomic DNA that comprises said genomic sequence; f) extending said support-bound selection primer to produce an extension product that contains a flanking sequence, wherein the flanking sequence is complementary to a sequence that flanks said genomic sequence in the genome; and g) annealing said extension product to an unextended member of said first and second populations of surface-bound oligonucleotides and amplifying said extension product on said substrate using unextended members of said first and second populations of surface-bound oligonucleotides, to produce a PCR product that comprises the flanking sequence.
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Specification