Methods of nucleic acid amplification and sequencing
First Claim
1. A method comprising the following steps:
- (1) forming at least one first nucleic acid template comprising at least one nucleic acid to be amplified, wherein said at least one nucleic acid contains (a) an oligonucleotide sequence Y and a means for attaching the at least one first nucleic acid template to a solid support at its 5′
end and (b) an oligonucleotide sequence Z at its 3′
end;
(2) mixing said at least one first nucleic acid template with one or more colony primers X, each of which carries a means for attaching one of the one or more colony primers X to the solid support at its 5′
end, in the presence of the solid support such that the 5′
ends of both the at least one first nucleic acid template and the one or more colony primers X bind to the solid support and resulting in at least one bound first template and one or more bound colony primers, wherein at least a subset of the one or more colony primers X can hybridize to the oligonucleotide sequence Z;
(3) performing one or more nucleic acid amplification reactions on the at least one bound first nucleic acid template, so that a first nucleic acid colony is generated, wherein the first nucleic acid colony comprises multiple copies of the at least one bound first nucleic acid template; and
(4) performing at least one step of sequence determination of one or more of the multiple copies of the at least one bound first nucleic acid template while the one or more of the multiple copies of the at least one bound first nucleic acid template is bound to the solid support.
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Accused Products
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) 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.
26 Citations
16 Claims
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1. A method comprising the following steps:
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(1) forming at least one first nucleic acid template comprising at least one nucleic acid to be amplified, wherein said at least one nucleic acid contains (a) an oligonucleotide sequence Y and a means for attaching the at least one first nucleic acid template to a solid support at its 5′
end and (b) an oligonucleotide sequence Z at its 3′
end;(2) mixing said at least one first nucleic acid template with one or more colony primers X, each of which carries a means for attaching one of the one or more colony primers X to the solid support at its 5′
end, in the presence of the solid support such that the 5′
ends of both the at least one first nucleic acid template and the one or more colony primers X bind to the solid support and resulting in at least one bound first template and one or more bound colony primers, wherein at least a subset of the one or more colony primers X can hybridize to the oligonucleotide sequence Z;(3) performing one or more nucleic acid amplification reactions on the at least one bound first nucleic acid template, so that a first nucleic acid colony is generated, wherein the first nucleic acid colony comprises multiple copies of the at least one bound first nucleic acid template; and (4) performing at least one step of sequence determination of one or more of the multiple copies of the at least one bound first nucleic acid template while the one or more of the multiple copies of the at least one bound first nucleic acid template is bound to the solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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Specification