EFFICIENT ARRAYS OF AMPLIFIED POLYNUCLEOTIDES
First Claim
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1. A method of making a random array of amplified polynucleotides, said method comprising the steps of:
- a) providing;
i) a surface, which comprises capture probes, wherein said capture probes have free 3′
ends, andii) a plurality of target polynucleotide concatemers disposed on said surface, wherein each concatemer is bound to capture probes at a specific position on said surface; and
b) extending said capture probes such that said concatemers disposed on said surface are amplified;
wherein the amplification products of said concatemers are attached to the surface at or near the specific position of the concatemer amplified, thereby making said random array of amplified polynucleotides.
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Abstract
The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are arrays of polynucleotides in which the polynucleotides have undergone multiple rounds of amplification in order to increase the strength of signals associated with single polynucleotide molecules.
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Citations
65 Claims
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1. A method of making a random array of amplified polynucleotides, said method comprising the steps of:
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a) providing; i) a surface, which comprises capture probes, wherein said capture probes have free 3′
ends, andii) a plurality of target polynucleotide concatemers disposed on said surface, wherein each concatemer is bound to capture probes at a specific position on said surface; and b) extending said capture probes such that said concatemers disposed on said surface are amplified; wherein the amplification products of said concatemers are attached to the surface at or near the specific position of the concatemer amplified, thereby making said random array of amplified polynucleotides. - View Dependent Claims (2, 7, 12, 13)
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- 3. The method of claim [0006], wherein said concatemers comprise multiple copies of individual target polynucleotides.
- 8. The method of claim [0006], wherein said surface is a planar surface that comprises discrete regions, wherein each discrete region comprises said capture probes attached thereto, and wherein at least a majority of said discrete spaced regions have amplification products of a single concatemer attached.
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14. A method of making a random array of target polynucleotides, said method comprising the steps of:
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a) providing a plurality of concatemers on a surface, wherein; i) said surface comprises capture probes, ii) each of said plurality of concatemers comprises multiple copies of a target polynucleotide and an adaptor; and iii) each concatemer is attached to a specific position on said surface through duplexes formed between said capture probes and said adaptors in the concatemer, wherein said duplexes comprise a recognition site for a nicking endonuclease; b) cleaving at least a portion of the plurality of concatemers with a nicking endonuclease to form cleavage products, wherein said cleavage products remain attached to said capture probes; c) circularizing said cleavage products on said capture probes; and d) extending said capture probes by rolling circle replication to create at least one copy of each circularized cleavage product; thereby making said random array of target polynucleotides. - View Dependent Claims (15, 16, 18)
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19. A method of making a random array of amplified target polynucleotides, said method comprising the steps of:
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a) providing a plurality of tailed concatemers; b) extending said tailed concatemers with a strand-displacing polymerase to form concatemer-extension product complexes; and c) disposing said concatemer-extension product complexes on a surface, wherein; i) said surface comprises capture probes, ii) a majority of said concatemer-extension product complexes from a single concatemer occupy a single region of said surface, and iii) said majority of said concatemer-extension product complexes is attached to said surface by one or more duplexes formed between said capture probes and said tail portions, thereby forming said random array of target polynucleotides. - View Dependent Claims (20, 21, 22, 23, 24, 25)
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26. A method of making a random array of target polynucleotides, said method comprising the steps of:
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a) combining under annealing conditions a plurality of dendrimers and a plurality of single stranded DNA circles, wherein; i) each of said plurality of single stranded DNA circles comprises a target polynucleotide and an adaptor, and ii) each of said plurality of dendrimers comprises a primer capable of annealing to said adaptors of said plurality of single stranded DNA circles, b) annealing said adaptors of said single stranded DNA circles to said primers of said plurality of dendrimers; c) extending said primers annealed with said adaptors with a strand-displacing polymerase to form a plurality of dendrimer-extension product complexes; and d) disposing said dendrimer-extension product complexes on a surface such that each of at least a majority of said plurality of dendrimer-extension product complexes occupies a separate region of said surface, thereby forming said random array of target polynucleotides. - View Dependent Claims (27, 28, 29, 30, 31)
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32. A method of forming a spatially-compact single stranded amplicon, said method comprising the steps of:
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a) combining under annealing conditions a dendrimer and a single stranded DNA circle, wherein; i) said single stranded DNA circle comprises a target polynucleotide and an adaptor, and ii) said dendrimer comprises a primer capable of annealing to said adaptor and at least one capture sequence identical to a portion of said single stranded DNA circle, b) extending said primer annealed to said adaptor with a strand-displacing polymerase to form a single-stranded amplicon, wherein said at least one capture sequence forms a duplex with a complementary portion of said single stranded amplicon, thereby forming said spatially-compact single stranded amplicon. - View Dependent Claims (33, 34, 35, 36)
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37. A method of forming a single-stranded amplicon comprising multiple target polynucleotide sequences, said method comprising the steps of:
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a) combining under annealing conditions a dendrimer and a plurality of single stranded DNA circles, wherein; i) each single stranded DNA circle comprises a target polynucleotide and an adaptor, and ii) said dendrimer comprises multiple sites complementary to adaptors on different DNA circles, b) extending said primer annealed to said adaptor with a strand-displacing polymerase to form a single stranded amplicon, wherein portions of each amplicon are complementary to target polynucleotides of different DNA circles, thereby forming said spatially compact single stranded amplicon comprising multiple target polynucleotide sequences. - View Dependent Claims (38)
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39. A method of forming a double stranded amplicon, the method comprising the steps of:
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a) providing a single stranded amplicon comprising a concatemer having multiple copies of a target polynucleotide and an adaptor; b) annealing primers to the adaptors of the single stranded amplicon; and c) extending the primers with a non-strand displacing polymerase so that substantially every annealed primer is extended to form an extension product that abuts the next annealed primer, thereby forming a double stranded amplicon. - View Dependent Claims (40)
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41. A method of making a random array of target polynucleotides, said method comprising the steps of:
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a) providing a support having a surface; b) combining in a reaction mixture first primer probes, beads comprising second primer probes on their surfaces, and a plurality of concatemers each comprising multiple copies of a target polynucleotide and an adaptor, said first primer and second primer probes being capable of amplifying in a polymerase chain reaction a portion of said concatemers; c) forming an emulsion with said reaction mixture so that aqueous compartments are formed that contain second primers, and no more than one bead and no more than one concatemer; d) conducting a polymerase chain reaction in said aqueous compartments so that portions of said concatemer are amplified on said beads; and e) disposing said beads from said emulsion onto said surface such that substantially every bead occupies a separate region of said surface, thereby forming said random array of target polynucleotides. - View Dependent Claims (42)
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43. A method of identifying a nucleotide sequence of a target polynucleotide, said method comprising:
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a) providing a random array comprising a plurality of concatemers disposed on a surface, wherein said concatemers comprise at least one fragment of said target polynucleotide, and wherein said concatemers have undergone at least one round of in situ amplification; b) hybridizing one or more probes from a first set of probes to said random array under conditions that permit formation of perfectly matched duplexes between said one or more probes from said first set of probes and complementary sequences on said concatemers; c) hybridizing one or more probes from a second set of probes to each random array under conditions that permit the formation of perfectly matched duplexes between said one or more probes from said second set of probes and complementary sequences on said concatemers; d) ligating probes from said first and second sets of probes which are hybridized to contiguous sites of said target concatemers; e) identifying sequences of said ligated probes, thereby generating a sequence read; f) repeating steps (b) through (e) a number of times to generate multiple sequence reads; g) assembling said multiple sequence reads, thereby identifying said nucleotide sequence of said target polynucleotide.
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44. A method of identifying a nucleotide sequence of a target polynucleotide, said method comprising:
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a) providing a random array comprising a plurality of concatemers disposed on a surface, wherein; i) said concatemers comprise at least one fragment of said target polynucleotide, ii) said concatemers further comprise at least one interspersed adaptors, wherein said at least one interspersed adaptor is adjacent to said at least one fragment, and iii) said concatemers have undergone at least one round of in situ amplification; b) identifying a sequence of at least a portion of said at least one fragment adjacent to said at least one interspersed adaptor; thereby identifying said nucleotide sequence of said target polynucleotide.
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45. A method of identifying a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
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a) providing a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises; i) a first target domain of said target sequence comprising a first set of target detection positions; ii) a first adaptor comprising a Type IIs endonuclease restriction site; iii) a second target domain of said target sequence comprising a second set of target detection positions; and iv) an interspersed adaptor comprising a Type IIs endonuclease restriction site; b) amplifying said plurality of concatemers; c) identifying said first nucleotide. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A random array of polynucleotides, wherein said random array comprises:
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a) a substrate having a surface; and b) a plurality of concatemer extension products disposed on said surface, wherein; i) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and ii) each of said plurality of concatemer extension products comprises at least two copies of a single-stranded concatemer comprising multiple copies of an identical polynucleotide sequence. - View Dependent Claims (58, 59, 60)
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61. A random array of target polynucleotides of unknown sequence, wherein said random array comprises:
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a) a substrate having a surface; and b) a plurality of concatemer extension products disposed on said surface, wherein; i) said plurality of concatemer extension products is formed from in situ amplification of immobilized concatemers, and ii) each of said plurality of concatemer extension products comprises at least two copies of a single-stranded concatemer comprising multiple copies of an identical target polynucleotide of unknown sequence. - View Dependent Claims (62, 63, 64)
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65. A kit for in situ amplification, wherein said kit comprises:
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a) a support having a surface which comprises first stage amplicons; b) adaptors and at least one ligase; c) at least one nicking endonuclease and reactants for a reaction using said at least one nicking endonuclease; and d) at least one polymerase and reactants for a synthesis reaction using said at least one is said polymerase.
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Specification