Device and method for amplifying target nucleic acid
A method for amplifying a target nucleic acid present in a sample includes introducing, via an entrance opening of a planar fluidic assembly, a sample containing one or more target nucleic acids into a flow channel of the planar fluidic assembly, the flow channel extending from the entrance opening to an exit vent and having a substantially uniform cross-section. A plurality of nucleic acid primers that are complementary to a portion of the one or more target nucleic acids are disposed at locations within and along the flow channel. The method further includes subjecting the sample introduced into the flow channel to a primer-based amplification reaction using the nucleic acid primers, wherein the primer-based amplification reaction produces amplified product of the one or more target nucleic acids, and retaining the amplified product at one or more of the locations within the flow channel during the primer-based amplification reaction. The method also includes detecting localized accumulation of the retained amplified product, the localized accumulation occurring at the one or more locations.
1. A method for amplifying a target nucleic acid present in a sample, the method comprising:
introducing, via an entrance opening of a planar fluidic assembly, a sample containing one or more target nucleic acids into a flow channel of the planar fluidic assembly, the flow channel extending from the entrance opening to an exit vent, the flow channel having a substantially uniform cross-section from the entrance opening to the exit vent, wherein a plurality of nucleic acid primers that are complementary to a portion of the one or more target nucleic acids are preloaded and retained at locations within and along the flow channel from the entrance opening to the exit vent;
subjecting the sample introduced into the flow channel to a primer-based amplification reaction using the nucleic acid primers, wherein the primer-based amplification reaction produces amplified product of the one or more target nucleic acids;
retaining the amplified product at one or more of the locations within the flow channel during the primer-based amplification reaction; and
detecting localized accumulation of the retained amplified product, the localized accumulation occurring at the one or more locations.
2. The method of claim 1 , wherein the plurality of nucleic acid primers are retained in a porous material at the locations.
3. The method of claim 1 , wherein the detecting comprises detecting fluorescence.
4. The method of claim 1 , wherein the flow channel comprises a plurality of flow channels.
5. The method of claim 2 , wherein the porous material-comprises a plurality of individual porous patches respectively positioned at the locations.
6. The method of claim 1 , wherein the fluidic assembly is a microscope slide assembly.
7. The method of claim 1 , further comprising retaining the sample in discrete reaction volumes in the flow channel, wherein the reaction volumes range from 1 picoliter to 1 microliter.
8. The method of claim 1 , further comprising protecting the sample and amplified product from evaporation during the primer-based amplification reaction.
9. The method of claim 1 , further comprising limiting diffusion of the sample and amplified product during the primer-based amplification reaction.
10. The method of claim 1 , wherein the primer-based amplification reaction comprises about 30-40 thermal cycles.
11. The method of claim 1 , wherein the primer-based amplification reaction initially amplifies a single molecule of the one or more target nucleic acids at one or more of the plurality of the locations.
12. The method of claim 1 , wherein the detecting comprises detecting light intensity indicative of 5′ exonuclease activity.