Methods for making nucleotide probes for sequencing and synthesis
First Claim
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1. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
- providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, and a bar code specific for a locus;
hybridizing the probes to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA;
ligating the probe to produce a closed circular molecule;
separating the closed circular molecule from the genomic DNA; and
ePCR amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying.
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Abstract
Compositions and methods for making a plurality of probes for analyzing a plurality of nucleic acid samples are provided. Compositions and methods for analyzing a plurality of nucleic acid samples to obtain sequence information in each nucleic acid sample are also provided.
37 Citations
24 Claims
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1. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, and a bar code specific for a locus; hybridizing the probes to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and ePCR amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (2, 3, 4, 5)
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6. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, and a bar code specific for a locus; hybridizing the probes to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and polony amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (7)
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8. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for a locus and a bar code specific for a patient; contacting the probes with genomic DNA to hybridize the probe in a circular manner to complementary genomic DNA; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and ePCR amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (9, 10)
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11. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for a locus and a bar code specific for a patient; contacting the probes with genomic DNA to hybridize the probe in a circular manner to complementary genomic DNA; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and polony amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (12)
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13. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, and a bar code specific for a locus; hybridizing the probes to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA with a one or more nucleotide gap between the ends of the circularized probe; polymerizing the extension of the probe in the presence of dATP, dCTP, dGTP or dTTP and a polymerase; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and ePCR amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (14, 15, 16, 17)
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18. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, and a bar code specific for a locus; hybridizing the probes to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA with a one or more nucleotide gap between the ends of the circularized probe; polymerizing the extension of the probe in the presence of dATP, dCTP, dGTP or dTTP and a polymerase; ligating the probe to produce a closed circular molecule; separating the closed circular molecule from the genomic DNA; and polony amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA. - View Dependent Claims (19)
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20. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for a locus and a bar code specific for a patient; contacting the probes with genomic DNA to hybridize the probe in a circular manner to complementary genomic DNA with a one or more nucleotide gap between the ends of the circularized probe; polymerizing the extension of the probe in the presence of dATP, dCTP, dGTP or dTTP and a polymerase; covalently attaching the extension to the end of the probe in the presence of a ligase to produce a closed circular molecule; and ePCR amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (21, 22)
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23. A method of analyzing a plurality of genomic DNA samples to obtain sequence information at one or more loci in each genomic DNA sample, the method comprising the steps of:
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providing one or more probes having two regions of homology to target genomic DNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for a locus and a bar code specific for a patient; contacting the probes with genomic DNA to hybridize the probe in a circular manner to complementary genomic DNA with a one or more nucleotide gap between the ends of the circularized probe; polymerizing the extension of the probe in the presence of dATP, dCTP, dGTP or dTTP and a polymerase; covalently attaching the extension to the end of the probe in the presence of a ligase to produce a closed circular molecule; and polony amplifying the closed circular molecule, wherein the closed circular molecule is separated from the genomic DNA prior to amplifying, and wherein the closed circular molecule is not cleaved prior to the amplifying. - View Dependent Claims (24)
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Specification