ENRICHMENT AND SEQUENCE ANALYSIS OF GENOMIC REGIONS
First Claim
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1. A method of reducing the genetic complexity of a population of nucleic acid molecules, the method comprising the steps of:
- (a) providing on a solid support single-stranded nucleic acid molecules of said population captured by specific hybridization to multiple, different oligonucleotide probes, wherein said nucleic acid molecules have an average size selected from the group consisting of about 100 to about 1000 nucleotide residues, about 250 to about 800 nucleotide residues and about 400 to about 600 nucleotide residues,(b) separating unbound and non-specifically hybridized nucleic acids from the captured molecules;
(c) eluting the captured molecules from the solid support, and(d) optionally repeating steps (a) to (c) for at least one further cycle with the eluted captured molecules.
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Abstract
The present invention provides novel methods for reducing the complexity of preferably a genomic sample for further analysis such as direct DNA sequencing, resequencing or SNP calling. The methods use pre-selected immobilized oligonucleotide probes to capture target nucleic acid molecules from a sample containing denatured, fragmented (genomic) nucleic acids for reducing the genetic complexity of the original population of nucleic acid molecules.
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Citations
49 Claims
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1. A method of reducing the genetic complexity of a population of nucleic acid molecules, the method comprising the steps of:
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(a) providing on a solid support single-stranded nucleic acid molecules of said population captured by specific hybridization to multiple, different oligonucleotide probes, wherein said nucleic acid molecules have an average size selected from the group consisting of about 100 to about 1000 nucleotide residues, about 250 to about 800 nucleotide residues and about 400 to about 600 nucleotide residues, (b) separating unbound and non-specifically hybridized nucleic acids from the captured molecules; (c) eluting the captured molecules from the solid support, and (d) optionally repeating steps (a) to (c) for at least one further cycle with the eluted captured molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for determining nucleic acid sequence information about at least one region of nucleic acid, the method comprising the steps of:
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1. reducing the genetic complexity of a population of nucleic acid molecules according to a method comprising the steps of; (a) providing on a solid support single-stranded nucleic acid molecules of said population captured by specific hybridization to multiple, different oligonucleotide probes, wherein said fragmented, denatured nucleic acid molecules have an average size selected from the group consisting of about 100 to about 1000 nucleotide residues, about 250 to about 800 nucleotide residues and about 400 to about 600 nucleotide residues, (b) separating unbound and non-specifically hybridized nucleic acids from the captured molecules; (c) eluting the captured molecules from the solid support, and (d) optionally repeating steps (a) to (c) for at least one further cycle with the eluted captured molecules; and 2. determining the nucleic acid sequence of the captured molecules. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method for detecting coding region variation relative to a reference genome, the method comprising the steps of:
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1. reducing the genetic complexity of a population of nucleic acid molecules according to a method comprising the steps of; (a) providing on a solid support single-stranded nucleic acid molecules of said population captured by specific hybridization to multiple, different oligonucleotide probes, wherein said fragmented, denatured nucleic acid molecules have an average size selected from the group consisting of about 100 to about 1000 nucleotide residues, about 250 to about 800 nucleotide residues and about 400 to about 600 nucleotide residues, (b) separating unbound and non-specifically hybridized nucleic acids from the captured molecules; (c) eluting the captured molecules from the solid support, and (d) optionally repeating steps (a) to (c) for at least one further cycle with the eluted captured molecules; 2. determining the nucleic acid sequence of the captured molecules, and 3. comparing the determined sequence to sequences in a database of the reference genome, in particular to sequences in a database of polymorphisms in the reference genome to identify variants from the reference genome. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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44. A kit comprising
double stranded adaptor molecules, and multiple, different oligonucleotide probes on a solid support, wherein said probes are selected from the group consisting of a plurality of probes that define a plurality of exons, introns or regulatory sequences from a plurality of genetic loci, a plurality of probes that define the complete sequence of at least one single genetic locus, a plurality of probes that define sites known to contain SNPs, and a plurality of probes that define an array designed to capture the complete sequence of at least one complete chromosome.
Specification