Microfluidic method for nucleic acid amplification
First Claim
1. An amplification device for target nucleic acids comprising a reaction chamber and at least one supply chamber, wherein amplicons complementary to a target nucleic acid segment of the target nucleic acid are generated in a reaction chamber within the device and form duplex amplicons between complementary amplicons, and wherein denaturation of the duplex amplicons is effected in the reaction chamber by a chemical denaturant or an electrostatic process wherein the device further comprises:
- an electric field generating device effective to create a field at a reaction chamber of the device such that the strands of amplicons in that chamber separate, wherein the amplicons are each formed from first primer and a second primer, where the first primers have one charge and the second primers have the opposite charge.
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Abstract
The nucleic acid amplification reactions of the present invention are conducted isothermally, using non-thermal denaturation procedures, such as occurs in chemical- or electrostatic-based denaturation. Embodiments are set forth for automated forms of the claimed procedures.
368 Citations
20 Claims
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1. An amplification device for target nucleic acids comprising a reaction chamber and at least one supply chamber, wherein amplicons complementary to a target nucleic acid segment of the target nucleic acid are generated in a reaction chamber within the device and form duplex amplicons between complementary amplicons, and wherein denaturation of the duplex amplicons is effected in the reaction chamber by a chemical denaturant or an electrostatic process wherein the device further comprises:
an electric field generating device effective to create a field at a reaction chamber of the device such that the strands of amplicons in that chamber separate, wherein the amplicons are each formed from first primer and a second primer, where the first primers have one charge and the second primers have the opposite charge. - View Dependent Claims (2, 3, 4)
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5. A method for amplification of a target nucleic acid, which target nucleic acid comprises a target nucleic acid segment, comprising, in a first cycle, the steps of:
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(A) providing one or more pairs of primers for creating extended primers, (B) denaturing the target nucleic acid by chemical means, (C) annealing the primers to the target nucleic acid to form a target-primer complex, (D) contacting the target-primer complex with polymerase to extend the primers of the target-primer complex, and (E) denaturing the target-primer complex of (D) by non-thermal means. - View Dependent Claims (6, 7, 17, 18, 19, 20)
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- 8. A microfluidic device for amplifying a nucleic acid, comprising a cassette comprised of at least one reaction flow-way having at least one reaction chamber connected to at least one supply chamber by a first fluid exchange channel, an electric field generating device effective to create a field at a reaction chamber of the device such that the strands of amplicons in that chamber separate, wherein the amplicons are each formed from first primer and a second primer, where the first primers have one charge and the second primers have the opposite charge.
Specification