Complexity management and analysis of genomic DNA
First Claim
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1. A method of analyzing a first nucleic acid sample comprising:
- providing said first nucleic acid sample;
obtaining a second nucleic acid sample by;
(a) fragmenting said first nucleic acid sample to produce fragments, ligating adaptor sequences to said fragments, amplifying at least some of said fragments, and isolating said amplified fragments;
or (b) fragmenting said first nucleic acid sample to produce fragments, denaturing said fragments, allowing at least some of said fragments to reanneal to form double stranded DNA sequences and removing said double stranded DNA sequences thus isolating said single stranded fragments;
or (c) amplifying said first nucleic acid sample by arbitrarily primed PCR to produce an amplification product and isolating said amplification product;
or (d) fragmenting said first nucleic acid sample to produce fragments, hybridizing said fragments to an oligonucleotide probe bound to a solid support, and isolating said hybridized fragments;
or (e) fragmenting said first nucleic acid sample to produce fragments, binding said fragments to a mismatch binding protein, and isolating said bound fragments;
providing a nucleic acid array;
hybridizing said second nucleic acid sample to said array; and
analyzing a hybridization pattern resulting from said hybridization.
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Abstract
The present invention provides for novel methods of sample preparation and analysis involving reproducibly reducing the complexity of a nucleic sample. The invention further provides for analysis of the above sample by hybridization to an array which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism. The invention further provides for novel methods of using a computer system to model enzymatic reactions in order to determine experimental conditions before conducting actual experiments.
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Citations
30 Claims
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1. A method of analyzing a first nucleic acid sample comprising:
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providing said first nucleic acid sample;
obtaining a second nucleic acid sample by;
(a) fragmenting said first nucleic acid sample to produce fragments, ligating adaptor sequences to said fragments, amplifying at least some of said fragments, and isolating said amplified fragments;
or(b) fragmenting said first nucleic acid sample to produce fragments, denaturing said fragments, allowing at least some of said fragments to reanneal to form double stranded DNA sequences and removing said double stranded DNA sequences thus isolating said single stranded fragments;
or(c) amplifying said first nucleic acid sample by arbitrarily primed PCR to produce an amplification product and isolating said amplification product;
or(d) fragmenting said first nucleic acid sample to produce fragments, hybridizing said fragments to an oligonucleotide probe bound to a solid support, and isolating said hybridized fragments;
or(e) fragmenting said first nucleic acid sample to produce fragments, binding said fragments to a mismatch binding protein, and isolating said bound fragments;
providing a nucleic acid array;
hybridizing said second nucleic acid sample to said array; and
analyzing a hybridization pattern resulting from said hybridization.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
binding oligonucleotide probes containing a desired SNP sequence to magnetic beads to form probe-bead complexes;
hybridizing said probe-bead complexes to said first nucleic acid sample;
exposing said first nucleic acid sample to a single strand DNA nuclease to remove single stranded DNA thereby obtaining only DNA duplexes;
ligating a double stranded adaptor sequence comprising a restriction enzyme site to said DNA duplexes;
digesting said DNA duplexes with a restriction enzyme to release the magnetic bead; and
isolating the duplexes.
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24. The method of claim 23 wherein said restriction enzyme is a Class IIs endonuclease.
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25. A method of screening for DNA sequence variations in an individual comprising:
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providing a first nucleic acid sample from said individual;
obtaining a second nucleic acid sample by;
(a) fragmenting said first nucleic acid sample to produce fragments, ligating adaptor sequences to said fragments, amplifying at least some of said fragments, and isolating said amplified fragments;
or(b) fragmenting said first nucleic acid sample to produce fragments, denaturing said fragments, allowing some of said fragments to reanneal to form double stranded DNA sequences and removing said double stranded DNA sequences thus isolating said single stranded fragments;
or(c) amplifying said first nucleic acid sample by arbitrarily primed PCR to produce an amplification product and isolating said amplification product;
or(d) fragmenting said first nucleic acid sample to produce fragments, hybridizing said fragments to an oligonucleotide probe bound to a solid support, and isolating said hybridized fragments;
or(e) fragmenting said first nucleic acid sample to produce fragments, binding said fragments to a mismatch binding protein, and isolating said bound fragments;
providing a nucleic acid array wherein said array comprises probes designed to interrogate for DNA sequence variations;
hybridizing said second nucleic acid sample to said array;
generating a hybridization pattern resulting from said hybridization; and
determining the presence or absence of DNA sequence variations in the individual based upon an analysis of the hybridization pattern. - View Dependent Claims (26, 27, 28)
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29. A method of screening for DNA sequence variations in a population of individuals comprising:
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providing a first nucleic acid sample from each of said individuals;
providing a second nucleic acid sample by;
(a) fragmenting said first nucleic acid sample to produce fragments, ligating adaptor sequences to said fragments, amplifying at least some of said fragments, and isolating said amplified fragments;
or(b) fragmenting said first nucleic acid sample to produce fragments, denaturing said fragments, allowing some of said fragments to reanneal to form double stranded DNA sequences and removing said double stranded DNA sequences thus isolating said single stranded fragments;
or(c) amplifying said first nucleic acid sample by arbitrarily primed PCR to produce an amplification product and isolating said amplification product;
or(d) fragmenting said first nucleic acid sample to produce fragments, hybridizing said fragments to an oligonucleotide probe bound to a solid support, and isolating said hybridized fragments;
or(e) fragmenting said first nucleic acid sample to produce fragments, binding said fragments to a mismatch binding protein, and isolating said bound fragments;
providing a plurality of identical nucleic acid arrays wherein said arrays comprise probes which are designed to interrogate for DNA sequence variations;
hybridizing each of said second nucleic acid samples to one of said plurality of identical arrays; and
generating a plurality of hybridization patterns resulting from said hybridizations; and
analyzing the hybridization patterns to determine the presence or absence of sequence variation in the population of individuals. - View Dependent Claims (30)
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Specification