Methods for Making Nucleotide Probes for Sequencing and Synthesis
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. CHURCH, Kun ZHANG, Joseph CHOU
Life of this patent
Estimating the term…
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.
Claims53
A method of selectively capturing a plurality of genomic sequences, the method comprising the steps of:
- providing a probe having two regions of homology to target genomic DNA at the ends of the probe, two common priming regions, and a restriction endonuclease recognition site;
- hybridizing the probe 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
- amplifying the closed circular molecule.
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
- amplifying the closed circular molecule.
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, 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;
- covalently attaching the extension to the end of the probe in the presence of a ligase to produce a closed circular molecule;
- and
- amplifying the circular molecule.
A method for enriching a probe pool for analyzing a plurality of genomic DNA regions in a genomic DNA sample, the method comprising the steps of:
- providing a plurality of probes each having two regions of degenerate homology to target genomic DNA at the ends of each probe, two PCR primer regions common to each probe, and a bar code specific for a genomic DNA region;
- hybridizing the probes to 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
- amplifying the closed circular molecule.
A method of analyzing a plurality of mRNA samples to obtain allele-specific quantitation at one or more alleles in each mRNA sample, the method comprising the steps of:
- providing one or more probes having two regions of homology to target mRNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for an allele, and a bar code specific for a patient;
- contacting the probes with mRNA to hybridize the probe in a circular manner to complementary mRNA;
- ligating the probe to produce a closed circular molecule;
- separating the closed circular molecule from the mRNA;
- and
- amplifying the closed circular molecule.
A method of analyzing a plurality of cDNA samples to obtain allele-specific quantitation at one or more alleles in each cDNA sample, the method comprising the steps of:
- providing one or more probes having two regions of homology to target cDNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for an allele, and a bar code specific for a patient;
- contacting the probes with cDNA to hybridize the probe in a circular manner to complementary cDNA;
- ligating the probe to produce a closed circular molecule;
- removing the closed circular molecule from the cDNA;
- and
- amplifying the closed circular molecule.
A method of selectively capturing a plurality of genomic sequences, the method comprising the steps of:
- providing a probe having two regions of homology to target genomic DNA at the ends of the probe, two common priming regions, and a restriction endonuclease recognition site;
- hybridizing the probe to immobilized genomic DNA such that the probe is hybridized in a circular manner to complementary genomic DNA;
- 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
- amplifying the closed circular molecule.
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 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
- amplifying the closed circular molecule.
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, 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
- amplifying the circular molecule.
A method for enriching a probe pool for analyzing a plurality of genomic DNA regions in a genomic DNA sample, the method comprising the steps of:
- providing a plurality of probes each having two regions of degenerate homology to target genomic DNA at the ends of each probe, two PCR primer regions common to each probe, and a bar code specific for a genomic DNA region;
- hybridizing the probes to 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
- amplifying the closed circular molecule.
A method of analyzing a plurality of mRNA samples to obtain allele-specific quantitation at one or more alleles in each mRNA sample, the method comprising the steps of:
- providing one or more probes having two regions of homology to target mRNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for an allele, and a bar code specific for a patient;
- contacting the probes with mRNA to hybridize the probe in a circular manner to complementary mRNA 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 mRNA;
- and
- amplifying the closed circular molecule.
A method of analyzing a plurality of cDNA samples to obtain allele-specific quantitation at one or more alleles in each cDNA sample, the method comprising the steps of:
- providing one or more probes having two regions of homology to target cDNA at the ends of the probe, two PCR primer regions common to all probes, a bar code specific for an allele, and a bar code specific for a patient;
- contacting the probes with cDNA to hybridize the probe in a circular manner to complementary cDNA 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;
- removing the closed circular molecule from the cDNA;
- and
- amplifying the closed circular molecule.
A method of making a plurality of probes comprising the steps of:
- providing linear, single stranded DNA encoding a plurality of probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two PCR primer regions common to all probes;
- amplifying the linear, single stranded DNA to form amplified, linear, double stranded DNA;
- converting the amplified, linear, double stranded DNA to linear, single stranded DNA;
- and
- releasing the plurality of probes from the linear, single stranded DNA.
Dependencies are inferred from the claim text.