Nucleic acid amplification
First Claim
1. A method for nucleic acid amplification, comprising:
- (a) forming a reaction mixture including a continuous liquid phase containing (i) a plurality of separate supports containing a first universal primer that does not include any target-specific sequence, (ii) a second universal primer in solution, (iii) a plurality of different polynucleotide templates containing both a first primer binding sequence and a second primer binding sequence, wherein the first primer binding sequence is complementary or identical to at least a portion of the first universal primer and the second primer binding sequence is complementary or identical to at least a portion of the second universal primer, and (iv) a polymerase; and
(b) forming two or more substantially monoclonal populations, each linked to a different support, by clonally amplifying, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the polynucleotide templates on different separate supports under isothermal amplification conditions using the first and second universal primers by partially denaturing and without compartmentalizing the polynucleotide templates.
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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.
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Citations
15 Claims
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1. A method for nucleic acid amplification, comprising:
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(a) forming a reaction mixture including a continuous liquid phase containing (i) a plurality of separate supports containing a first universal primer that does not include any target-specific sequence, (ii) a second universal primer in solution, (iii) a plurality of different polynucleotide templates containing both a first primer binding sequence and a second primer binding sequence, wherein the first primer binding sequence is complementary or identical to at least a portion of the first universal primer and the second primer binding sequence is complementary or identical to at least a portion of the second universal primer, and (iv) a polymerase; and (b) forming two or more substantially monoclonal populations, each linked to a different support, by clonally amplifying, within the same reaction mixture of step (a) in the continuous liquid phase, at least two of the polynucleotide templates on different separate supports under isothermal amplification conditions using the first and second universal primers by partially denaturing and without compartmentalizing the polynucleotide templates. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification