Methods and compositions for whole genome amplification and genotyping

• US 20050059048A1
• Filed: 06/17/2004
• Published: 03/17/2005
• Est. Priority Date: 06/20/2003
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Gunderson, Kevin, Steemers, Frank
• Granted Patent: US 7,670,810 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide

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.

Claims

1-77. -77. (cancelled).

78. A method, comprising

(a) providing a plurality of genome fragments,

wherein said plurality of genome fragments comprises at least 100 ug of DNA comprising a complexity of at least 1 Gigabases;

(b) contacting said plurality of genome fragments with a plurality of different immobilized nucleic acid probes,

wherein at least 500 of said different nucleic acid probes hybridize with genome fragments to form probe-fragment hybrids; and

(c) detecting typable loci of said probe-fragment hybrids.

79. The method of claim 78, wherein said plurality of different immobilized nucleic acid probes comprises an array of said probes attached to a surface.

80. The method of claim 78, wherein said different immobilized nucleic acid probes are attached to particles.

81. The method of claim 80, wherein each of said particles is attached to a single type of nucleic acid probe.

82. The method of claim 80, wherein said particles are attached to a substrate.

83. The method of claim 78, wherein said plurality of genome fragments comprises a concentration of least 1 ug/ul of DNA.

84. The method of claim 78, wherein at least 100,000 of said different nucleic acid probes hybridize with genome fragments to form probe-fragment hybrids.

85. The method of claim 78, wherein said detecting comprises modifying said immobilized probes while hybridized to said genome fragments.

86. The method of claim 85, wherein said modifying comprises addition of a nucleotide or nucleotide analog by a polymerase.

87. The method of claim 85, wherein said modifying comprises ligation of probes to said immobilized nucleic acid probes.

88. The method of claim 85, wherein said modifying comprises cleavage of said immobilized nucleic acid probes.

89. The method of claim 85, further comprising contacting said probes with a single-stranded nucleic acid binding protein.

90. The method of claim 78, wherein said providing comprises representationally amplifying a native genome.

91. The method of claim 78, wherein said providing comprises linker adapter-PCR.

92. The method of claim 78, wherein said providing comprises random primer amplification.

93. The method of claim 92, comprising amplification using a polymerase having low processivity.

94. The method of claim 78, wherein said providing comprises treating genomic DNA with an endonuclease.

95. The method of claim 94, wherein said endonucleases comprises DNAse I.

96. The method of claim 78, wherein said providing comprises treating genome fragments with an endonuclease.

97. The method of claim 96, wherein said endonucleases comprises DNAse I.

98. The method of claim 78, further comprising modifying said immobilized probes while hybridized to said genome fragments, thereby forming modified immobilized probes.

99. The method of claim 98, further comprising exposing said modified immobilized probes to denaturing conditions prior to said detecting, thereby removing said genome fragments.

100. The method of claim 78, further comprising modifying said immobilized probe-fragment hybrids by addition of a detection moiety to the probe, thereby forming affinity ligand-labeled probes.

101. The method of claim 100, further comprising contacting said affinity ligand-labeled probes with a receptor and an amplification reagent,

wherein said receptor has one or more sites capable of binding said ligand, and wherein said amplification reagent has affinity for said receptor,

whereby multimeric complexes form between said affinity ligand-labeled probes, said receptor and said amplification reagent.

102. The method of claim 101, wherein said detecting comprises detecting said multimeric complexes.

103. The method of claim 78, wherein step (a) is carried out in a capillary gap flow cell.

104. A method, comprising

(a) providing a plurality of genome fragments,

wherein said plurality of genome fragments comprises a concentration of at least 1 ug/ul of DNA comprising a complexity of at least 1 Gigabases;

(b) contacting said plurality of genome fragments with a plurality of different immobilized nucleic acid probes,

wherein at least 500 of said different nucleic acid probes hybridize with genome fragments to form probe-fragment hybrids; and

(c) detecting typable loci of said probe-fragment hybrids.

105. The method of claim 104, wherein said plurality of different immobilized nucleic acid probes comprises an array of said probes attached to a surface.

106. The method of claim 104, wherein said different immobilized nucleic acid probes are attached to particles.

107. The method of claim 106, wherein each of said particles is attached to a single type of nucleic acid probe.

108. The method of claim 106, wherein said particles are attached to a substrate.

109. The method of claim 104, wherein at least 100,000 of said different nucleic acid probes hybridize with genome fragments to form probe-fragment hybrids.

110. The method of claim 104, wherein said detecting comprises modifying said immobilized probes while hybridized to said genome fragments.

111. The method of claim 110, wherein said modifying comprises addition of a nucleotide or nucleotide analog by a polymerase.

112. The method of claim 110, wherein said modifying comprises ligation of probes to said immobilized nucleic acid probes.

113. The method of claim 110, wherein said modifying comprises cleavage of said immobilized nucleic acid probes.

114. The method of claim 110, further comprising contacting said probes with a single-stranded nucleic acid binding protein.

115. The method of claim 104, wherein said providing comprises representationally amplifying a native genome.

116. The method of claim 104, wherein said providing comprises linker adapter-PCR.

117. The method of claim 104, wherein said providing comprises random primer amplification.

118. The method of claim 117, comprising amplification using a polymerase having low processivity.

119. The method of claim 104, wherein said providing comprises treating genomic DNA with an endonuclease.

120. The method of claim 119, wherein said endonucleases comprises DNAse I.

121. The method of claim 104, wherein said providing comprises treating genome fragments with an endonuclease.

122. The method of claim 121, wherein said endonucleases comprises DNAse I.

123. The method of claim 104, further comprising modifying said immobilized probes while hybridized to said genome fragments, thereby forming modified immobilized probes.

124. The method of claim 123, further comprising exposing said modified immobilized probes to denaturing conditions prior to said detecting, thereby removing said genome fragments.

125. The method of claim 104, further comprising modifying said immobilized probe-fragment hybrids by addition of a detection moiety to the probe, thereby forming affinity ligand-labeled probes.

126. The method of claim 125, further comprising contacting said affinity ligand-labeled probes with a receptor and an amplification reagent,

wherein said receptor has one or more sites capable of binding said ligand, and wherein said amplification reagent has affinity for said receptor,

whereby multimeric complexes form between said affinity ligand-labeled probes, said receptor and said amplification reagent.

127. The method of claim 126, wherein said detecting comprises detecting said multimeric complexes.

128. The method of claim 104, wherein step (a) is carried out in a capillary gap flow cell.