Methods of nucleic acid amplification and sequencing
First Claim
1. A method for amplification of at least one nucleic acid, comprising the following steps:
- (1) forming at least one nucleic acid template comprising the at least one nucleic acid to be amplified, wherein the at least one nucleic acid contains an oligonucleotide sequence Y at the 5′
end and an oligonucleotide sequence Z at the 3′
end, and the at least one nucleic acid carries a means for immobilizing the at least one nucleic acid to a solid support at the 5′
end;
(2) mixing the at least one nucleic acid template, in the presence of the solid support, with one or more colony primers X, each of which can hybridize to the oligonucleotide sequence Z and carries a means for immobilizing the colony primer to the solid support at the 5′
end, whereby the 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to the solid support;
wherein said 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to said solid support such that they cannot be removed by washing with water or aqueous buffer under DNA-denaturing conditions; and
(3) performing one or more nucleic acid amplification reactions on the immobilized nucleic acid template, so that nucleic acid colonies are generated.
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Abstract
Methods for amplification and sequencing of at least one nucleic acid comprising the following steps: (1) forming at least one nucleic acid template comprising the nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) contains at the 5′ end an oligonucleotide sequence Y and at the 3′ end an oligonucleotide sequence Z and, in addition, the nucleic acid(s) carry at the 5′ end a means for attaching the nucleic acid(s) to a solid support; (2) mixing said nucleic acid template(s) with one or more colony primers X, which can hybridize to the oligonucleotide sequence Z and carries at the 5′ end a means for attaching the colony primers to a solid support, in the presence of a solid support so that the 5′ ends of both the nucleic acid template and the colony primers bind to the solid support; (3) performing one or more nucleic acid amplification reactions on the bound template(s), so that nucleic acid colonies are generated and optionally, performing at least one step of sequence determination of one or more of the nucleic acid colonies generated. Solid supports, kits and apparatus for use in these methods.
710 Citations
20 Claims
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1. A method for amplification of at least one nucleic acid, comprising the following steps:
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(1) forming at least one nucleic acid template comprising the at least one nucleic acid to be amplified, wherein the at least one nucleic acid contains an oligonucleotide sequence Y at the 5′
end and an oligonucleotide sequence Z at the 3′
end, and the at least one nucleic acid carries a means for immobilizing the at least one nucleic acid to a solid support at the 5′
end;(2) mixing the at least one nucleic acid template, in the presence of the solid support, with one or more colony primers X, each of which can hybridize to the oligonucleotide sequence Z and carries a means for immobilizing the colony primer to the solid support at the 5′
end, whereby the 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to the solid support;wherein said 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to said solid support such that they cannot be removed by washing with water or aqueous buffer under DNA-denaturing conditions; and(3) performing one or more nucleic acid amplification reactions on the immobilized nucleic acid template, so that nucleic acid colonies are generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for amplification of at least one nucleic acid, comprising the following steps:
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(1) forming at least one nucleic acid template comprising the at least one nucleic acid to be amplified, wherein the at least one nucleic acid contains an oligonucleotide sequence Y at the 5′
end and an oligonucleotide sequence Z at the 3′
end, and the at least one nucleic acid carries a means for immobilizing the at least one nucleic acid to a solid support at the 5′
end;(2) mixing the at least one nucleic acid template, in the presence of the solid support, with one or more degenerate colony primers X, each of which can hybridize to the oligonucleotide sequence Z and carries a means for immobilizing the colony primer to the solid support at the 5′
end, whereby the 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to the solid support;
wherein said 5′
ends of both the at least one nucleic acid template and the colony primers are immobilized to said solid support such that they cannot be removed by washing with water or aqueous buffer under DNA-denaturing conditions, and(3) performing one or more nucleic acid amplification reactions on the immobilized nucleic acid template, so that nucleic acid colonies are generated. - View Dependent Claims (20)
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Specification