Methods and compositions for whole genome amplification and genotyping
First Claim
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1. A method for detecting at least 10,000 typable loci comprising single-nucleotide polymorphisms (SNPs) of genome fragments, comprising:
- (a) providing a plurality of genome fragments of a whole genome,wherein said providing comprises representationally amplifying a whole, native genome, andwherein said plurality of genome fragments comprises a concentration of at least 1 μ
g/μ
l of DNA;
(b) contacting said plurality of genome fragments with an array of at least 10,000 predetermined nucleic acid probes immobilized to a substrate,wherein said probes comprise a region of at most 100 nucleotides in length that is complementary to typable loci of a genome, andwherein said probes hybridize with genome fragments to form probe-fragment hybrids;
(c) modifying the probe of a probe-fragment hybrid while hybridized to said genome fragments by adding a detectable nucleotide or nucleotide analog by a polymerase;
(d) exposing said modified probes to denaturing conditions, thereby removing hybridized genome fragments; and
(e) detecting said modified probes, thereby detecting at least 10,000 typable loci comprising SNPs.
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Abstract
This invention provides methods of amplifying genomic DNA to obtain an amplified representative population of genome fragments. Methods are further provided for obtaining amplified genomic DNA representations of a desired complexity. The invention further provides methods for simultaneously detecting large numbers of typable loci for an amplified representative population of genome fragments. Accordingly the methods can be used to genotype individuals on a genome-wide scale.
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Citations
26 Claims
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1. A method for detecting at least 10,000 typable loci comprising single-nucleotide polymorphisms (SNPs) of genome fragments, comprising:
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(a) providing a plurality of genome fragments of a whole genome, wherein said providing comprises representationally amplifying a whole, native genome, and wherein said plurality of genome fragments comprises a concentration of at least 1 μ
g/μ
l of DNA;(b) contacting said plurality of genome fragments with an array of at least 10,000 predetermined nucleic acid probes immobilized to a substrate, wherein said probes comprise a region of at most 100 nucleotides in length that is complementary to typable loci of a genome, and wherein said probes hybridize with genome fragments to form probe-fragment hybrids; (c) modifying the probe of a probe-fragment hybrid while hybridized to said genome fragments by adding a detectable nucleotide or nucleotide analog by a polymerase; (d) exposing said modified probes to denaturing conditions, thereby removing hybridized genome fragments; and (e) detecting said modified probes, thereby detecting at least 10,000 typable loci comprising SNPs. - 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, 25, 26)
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Specification