Nucleic acid amplification
First Claim
1. A method for nucleic acid amplification, comprising:
- (a) forming a reaction mixture by contacting, within a continuous liquid phase under nucleic acid synthesis conditions;
a plurality of different target polynucleotide templates in solution, each containing a first primer binding sequence and a second primer binding sequence, wherein the support contains only one type of primer;
a support containing multiple copies of a first universal primer that does not include any target-specific sequence and includes a sequence that is complementary or identical to the first primer-binding sequence, and the support does not include a primer containing a sequence that is complementary or identical to the first primer binding sequence, wherein the support contains only one type of primer;
multiple copies of a second universal primer in solution, wherein the second universal primer includes a sequence that complementary or identical to the second primer-binding sequence and is optionally pre-hybridized to one or more of the target polynucleotide templates; and
a polymerase; and
(b) forming two or more substantially monoclonal populations that are linked to the support, by clonally amplifying, by partially denaturing and without first compartmentalizing, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the target polynucleotide templates on the support using the first and second universal primers under isothermal amplification conditions.
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Accused Products
Abstract
In some embodiments, the present teachings provide methods for nucleic acid amplification, comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, methods for nucleic acid amplification include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, methods for nucleic acid amplification include amplifying without fully denaturing the nucleic acid that is amplified. In some embodiments, the methods for nucleic acid amplification employ an enzyme that catalyzes homologous recombination and a polymerase. In some embodiments, methods for nucleic acid amplification can be conducted in a single reaction vessel. In some embodiments, methods for nucleic acid amplification can be conducted in a single continuous liquid phase of a reaction mixture, without need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, methods for nucleic acid amplification comprise a amplifying at least one polynucleotide onto a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer can include a sieving agent and/or a diffusion-reducing agent.
89 Citations
17 Claims
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1. A method for nucleic acid amplification, comprising:
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(a) forming a reaction mixture by contacting, within a continuous liquid phase under nucleic acid synthesis conditions; a plurality of different target polynucleotide templates in solution, each containing a first primer binding sequence and a second primer binding sequence, wherein the support contains only one type of primer; a support containing multiple copies of a first universal primer that does not include any target-specific sequence and includes a sequence that is complementary or identical to the first primer-binding sequence, and the support does not include a primer containing a sequence that is complementary or identical to the first primer binding sequence, wherein the support contains only one type of primer; multiple copies of a second universal primer in solution, wherein the second universal primer includes a sequence that complementary or identical to the second primer-binding sequence and is optionally pre-hybridized to one or more of the target polynucleotide templates; and a polymerase; and (b) forming two or more substantially monoclonal populations that are linked to the support, by clonally amplifying, by partially denaturing and without first compartmentalizing, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the target polynucleotide templates on the support using the first and second universal primers under isothermal amplification conditions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification