Methods and compositions for whole genome amplification and genotyping
First Claim
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1. A method of detecting typable loci of a genome, comprising the steps of:
- (a) providing an amplified representative population of genome fragments comprising said typable loci, wherein said population comprises a high complexity representation;
(b) contacting said genome fragments with a plurality of nucleic acid probes having sequences corresponding to said typable loci under conditions wherein probe-fragment hybrids are formed, wherein said probes are at most 125 nucleotides in length; and
(c) detecting typable loci of said probe-fragment hybrids.
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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.
241 Citations
127 Claims
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1. A method of detecting typable loci of a genome, comprising the steps of:
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(a) providing an amplified representative population of genome fragments comprising said typable loci, wherein said population comprises a high complexity representation;
(b) contacting said genome fragments with a plurality of nucleic acid probes having sequences corresponding to said typable loci under conditions wherein probe-fragment hybrids are formed, wherein said probes are at most 125 nucleotides in length; and
(c) detecting typable loci of said probe-fragment hybrids. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 34, 35)
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18. A method of detecting typable loci of a genome, comprising the steps of:
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(a) providing an amplified representative population of genome fragments comprising said typable loci;
(b) contacting said genome fragments with a plurality of nucleic acid probes having sequences corresponding to said typable loci under conditions wherein probe-fragment hybrids are formed; and
(c) directly detecting typable loci of said probe-fragment hybrids. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 36)
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37. A method of detecting typable loci of a genome, comprising the steps of:
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(a) providing an amplified representative population of genome fragments comprising said typable loci;
(b) contacting said genome fragments with a plurality of immobilized nucleic acid probes having sequences corresponding to said typable loci under conditions wherein immobilized probe-fragment hybrids are formed;
(c) modifying said immobilized probe-fragment hybrids; and
(d) detecting a probe or fragment modified in step (c), thereby detecting said typable loci of said genome. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54)
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55. A method of amplifying genomic DNA, comprising the steps of:
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(a) providing isolated double stranded genomic DNA;
(b) contacting said double stranded genomic DNA with a nicking agent, thereby producing nicked double stranded genomic DNA; and
(c) contacting said nicked double stranded genomic DNA with a strand displacing polymerase and a plurality of primers, wherein said genomic DNA is amplified. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62, 63)
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64. A method for detecting typable loci of a genome, comprising the steps of
(a) in vitro transcribing a population of amplified genome fragments, thereby obtaining genomic RNA fragments; -
(b) hybridizing said genomic RNA fragments with a plurality of nucleic acid probes having sequences corresponding to said typable loci, thereby forming a plurality of RNA fragment-probe hybrids; and
(c) detecting typable loci of said RNA fragment-probe hybrids. - View Dependent Claims (65, 66, 67, 68, 69, 70, 71, 72)
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73. A method of producing a reduced complexity, locus-specific, amplified representative population of genome fragments, comprising the steps of
(a) replicating a native genome with a plurality of random primers, thereby producing an amplified representative population of genome fragments; -
(b) replicating a sub-population of said amplified representative population of genome fragments with a plurality of different locus-specific primers, thereby producing a locus-specific, amplified representative population of genome fragments; and
(c) isolating said sub-population, thereby producing a reduced complexity, locus-specific, amplified representative population of genome fragments. - View Dependent Claims (74, 75, 76, 77)
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78. A method of detecting typable loci of a genome, comprising the steps of:
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(a) representationally amplifying a native genome, wherein an amplified representative population of genome fragments comprising said typable loci is produced under isothermal conditions;
(b) contacting said genome fragments with a plurality of different nucleic acid probes having sequences corresponding to said typable loci under conditions wherein probe-fragment hybrids are formed; and
(c) detecting typable loci of said probe-fragment hybrids. - View Dependent Claims (79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107)
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108. A method of detecting typable loci of a genome, comprising the steps of:
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(a) representationally amplifying a native genome, wherein an amplified representative population of genome fragments comprising said typable loci is produced under isothermal conditions, wherein said genome fragments comprise a base exogenous to native DNA at 1% to 10% of the positions for A, T, G or C;
(b) treating said genome fragments with a reagent under conditions wherein said genome fragments comprising said base exogenous to native DNA are cleaved, thereby producing smaller genome fragments;
(c) contacting said smaller genome fragments with a plurality of different nucleic acid probes having sequences corresponding to said typable loci under conditions wherein hybrids are formed between said probes and said smaller genome fragments; and
(d) detecting typable loci of said hybrids. - View Dependent Claims (109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127)
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Specification