Random array DNA analysis by hybridization
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Abstract
The invention relates to methods and devices for analyzing single molecules, i.e. nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.
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Citations
59 Claims
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1-23. -23. (canceled)
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24. A method to analyze nucleic acids comprising:
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(a) hybridizing a population of oligonucleotide probes comprising the same region By to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (b) collecting a signal produced by multiple consecutive hybridization of oligonucleotide probes to the target nucleic acids on the array; and (c) analyzing said signal. - View Dependent Claims (25, 26)
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27. A method to analyze target nucleic acids comprising:
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(a) hybridizing a population of oligonucleotide probes comprising the same region By to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (b) ligating at least two oligonucleotide probes that are hybridized to the target nucleic acid molecules on the array; (c) collecting a signal produced by the multiple consecutive hybridization of oligonucleotides molecules and probe ligation; and (d) analyzing said signal. - View Dependent Claims (28, 29)
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30. A method of determining the sequence of a target nucleic acid, comprising the steps of:
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(a) hybridizing a first set of detectably-labeled oligonucleotide probes to a random array of nucleic acids generated from the target nucleic acid under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (b) hybridizing a second set of detectably-labeled oligonucleotide probes to said target nucleic acids on the array under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (c) ligating at least one probe from each set that are hybridized to said target nucleic acids on the array; and (d) detecting and analyzing said ligated probes; wherein a sequence of the target molecule is assembled by ordering overlapping probe sequences that hybridize to the target molecule. - View Dependent Claims (31, 32, 33)
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34. A method to analyze nucleic acids comprising:
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(a) hybridizing a first set of detectably labeled oligonucleotide probes to an array of target nucleic acids under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (b) hybridizing a second set of detectably labeled oligonucleotide probes to the array under reaction conditions optimized for multiple consecutive hybridization to full-match sequences; (c) ligating at least two detectably labeled probes that are hybridized to nucleic acids on the array; and (d) detecting a fluorescence resonance energy transfer (FRET) signal between said labeled probes. - View Dependent Claims (35, 36, 37, 38)
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39. A composition or mixture comprising a first set of probes associated with a FRET acceptor molecule, a second set of probes associated with a FRET donor molecule, and a ligating molecule.
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40. A method of amplifying a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:
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(a) binding an invader oligonucleotide to one end of the target nucleic acid; (b) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid; (c) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase; (d) hybridizing a second primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (b); (e) creating a new double stranded nucleic acid using the strand displacement polymerase; and (f) repeating steps (a)-(e). - View Dependent Claims (41)
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42. A method of amplifying a target DNA on a random array of nucleic acids, comprising the steps of:
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(a) providing an invader oligonucleotide and a first primer complementary to a portion of the invader oligonucleotide; (b) binding the invader oligonucleotide to one end of the target DNA; (c) hybridizing the first primer oligonucleotide to a single-stranded site on the target nucleic acid; (d) initiating primer extension and displacement of one strand of the DNA using a strand displacement polymerase; (e) hybridizing a second primer oligonucleotide to a single-stranded site of the target DNA on the opposite strand to the single-stranded site in step (c); (f) creating a new copy of the target DNA using the polymerase; and (g) repeating steps (b) through (f). - View Dependent Claims (43)
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44. A method of isothermal amplification of a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:
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(a) binding an invader oligonucleotide to the 5′
end of the target nucleic acid;(b) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid; (c) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase; (d) hybridizing a second primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (b); (e) creating a new double stranded nucleic acid using the strand displacement polymerase; and (f) repeating steps (a)-(e). - View Dependent Claims (45, 46)
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47. A method of isothermal amplification of a double-stranded target nucleic acid on a random array of nucleic acids, comprising the steps of:
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(a) adding a double stranded nucleic acid fragment with low duplex stability to the 5′
end of the target nucleic acid;(b) binding an invader oligonucleotide to the 5′
end of the target nucleic acid;(c) hybridizing a first primer oligonucleotide to a single-stranded site on the target nucleic acid; (d) initiating primer extension and displacement of one nucleic acid strand by a strand displacement polymerase; (e) hybridizing a second primer oligonucleotide to a single stranded site of the target nucleic acid on the opposite strand to the single-stranded site in step (c); (f) creating a new double stranded nucleic acid using the strand displacement polymerase; and (g) repeating steps (b)-(f). - View Dependent Claims (48)
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49. A method to analyze nucleic acids comprising:
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(a) providing a random array of target nucleic acids, wherein the target nucleic acids are generated from multiple sources, and wherein each nucleic acid is tagged with an identifier of the particular source of the nucleic acid; (b) hybridizing a population of oligonucleotide probes comprising the same region By to the array of target nucleic acids under reaction conditions optimized for hybridization to full-match sequences; (c) collecting a signal produced by hybridization of oligonucleotide probes to the target nucleic acids on the array; and (d) analyzing said signal; wherein nucleic acids from a particular source are recognized by the assigned tag sequence. - View Dependent Claims (50)
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51. A method of sequencing a plurality of nucleic acids from multiple sample sources, the method comprising:
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(a) providing a random array of nucleic acids, wherein the nucleic acids are generated from multiple sources, and wherein each nucleic acid is tagged with identifier of the particular source of each nucleic acid; (b) hybridizing a first set of detectably-labeled oligonucleotide probes to a random array of nucleic acids generated from the target nucleic acid under reaction conditions optimized for hybridization to full-match sequences; (c) hybridizing a second set of detectably-labeled oligonucleotide probes to said target nucleic acids on the array under reaction conditions optimized for hybridization to full-match sequences; (d) ligating at least one probe from each set that are hybridized to said target nucleic acids on the array; and (e) detecting and analyzing said ligated probes; wherein a sequence of each nucleic acid is identified by probe sequences that hybridize to the target molecule, and wherein nucleic acids from a particular source are recognized by the assigned tag sequence. - View Dependent Claims (52)
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53. An assay for detecting a sequence in a target DNA fragment, comprising:
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(a) providing a support comprising a plurality of target DNA fragments attached thereto; (b) hybridizing two or more probes to target DNA fragments on said support; (c) ligating the probes on said target nucleic acid that are hybridized to adjacent sites on a target DNA fragment to form a ligated probe pair; (d) removing any probes that are not ligated to another probe on a target DNA fragment; and (e) detecting ligated probe pairs hybridized to the target DNA fragment; wherein detection of a ligated probe pair on a target DNA fragment is indicative of a sequence in the a target DNA fragment. - View Dependent Claims (54)
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55. An assay for detecting a sequence in a target DNA fragment, comprising:
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(a) providing a support comprising a plurality of target DNA fragments attached thereto; (b) hybridizing one or more probes to target DNA fragments, wherein said probes comprise a donor fluorophore for fluorescence resonance energy transfer (FRET) detection; (c) hybridizing one or more probes to target DNA fragments, wherein said probes comprise a acceptor fluorophore for FRET detection; (d) ligating probes on said target nucleic acid that are hybridized to adjacent sites on a target DNA fragment to form a ligated probe pair; (e) removing any probes that are not ligated to another probe on a target DNA fragment; and (f) detecting a fluorescence resonance energy transfer (FRET) signal between said ligated probes; wherein detection of a FRET signal generated by a ligated probe pair on a target DNA fragment is indicative of a sequence in the a target DNA fragment. - View Dependent Claims (56)
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57. A sequence identification system, comprising:
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(a) A random array of single molecule DNA fragments; (b) A set of fluorescently tagged probe pairs from informative probe pools; and (c) a camera for detection of hybridization/ligation events on the array; wherein the system is designed to allowing testing of ligation of a given number of informative bases using multiple different probes capable of ligation with probes from the informative probe pools when hybridized contiguously on a target polynucleotide. - View Dependent Claims (58, 59)
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Specification