Device and method for amplifying target nucleic acid

• US 9,506,105 B2
• Filed: 08/11/2014
• Issued: 11/29/2016
• Est. Priority Date: 04/17/1997
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America), Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: United States Department Of Health And Human Services (Government of the United States of America), Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Brown, James F., Silver, Jonathan E.
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US14/456,592
• Publication Number:
  US 20150010911A1
• Time in Patent Office: 841 Days
• Field of Search: 435/6.1
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

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.

Claims

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.