METHOD AND DEVICE FOR DETECTING THE PRESENCE OF A SINGLE TARGET NUCLEIC ACID IN A SAMPLE

• US 20090035759A1
• Filed: 08/13/2007
• Published: 02/05/2009
• Est. Priority Date: 04/17/1997
• Status: Active Grant

• 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: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: Silver, Jonathan E., BROWN, James F.
• Granted Patent: US 8,859,204 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 comprises loading a sample portion into a sample chamber which comprises means for minimizing diffusion of the sample portion, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a sample portion and a sample chamber comprising means for minimizing diffusion of the sample portion. Also, a microfluidic device comprising a sample chamber and an amplification targeting reagent positioned in the first sample chamber.

Claims

1. A method for detecting whether at least one molecule of a target nucleic acid is:

present in a first sample portion, said method comprising;

loading a first sample portion into a first sample chamber, said first sample portion comprising at least part of a sample, whereby if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification, said first sample chamber comprising means for minimizing diffusion of said first sample portion;

subjecting said first sample portion in said first sample chamber to at least a first amplification step; and

then

determining whether said first sample portion contains at least one molecule of said target nucleic acid.

2. A method as recited in claim 1, wherein said means for minimizing diffusion of said first sample portion comprises at least one physical restraint on diffusion.

3. A method as recited in claim 1, wherein said means for minimizing diffusion of said first sample portion comprises at least one chemical restraint on diffusion.

4. A method as recited in claim 1, wherein said means for minimizing diffusion of said first sample portion comprises at least one feature selected from among flow restriction means, glass beads, gels, absorbent particles and electrophoretic means.

5. A method as recited in claim 1, wherein said loading of said first sample portion is carried out using at least centrifugal force.

6. A method as recited in claim 1, wherein said first sample portion is loaded into said first sample chamber through at least one ink jet.

7. A method as recited in claim 1, wherein said loading of said first sample portion is carried out using at least pressure.

8. A method as recited in claim 1, wherein said loading of said first sample portion is carried out using at least a vacuum.

9. A method as recited in claim 1, wherein said loading of said first sample portion is carried out using at least capillary action.

10. A microfluidic device comprising:

a first sample chamber; and

a first sample portion, said first sample portion being positioned in said first sample chamber, said first sample chamber comprising means for minimizing diffusion of said first sample portion, whereby if said first sample portion contains at least a single molecule of a target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

11. A microfluidic device as recited in claim 10, wherein said means for minimizing diffusion of said first sample portion comprises at least one physical restraint on diffusion.

12. A microfluidic device as recited in claim 10, wherein said means for minimizing diffusion of said first sample portion comprises at least one chemical restraint on diffusion.

13. A microfluidic device as recited in claim 10, wherein said means for minimizing diffusion of said first sample portion comprises at least one feature selected from among flow restriction means, glass beads, gels, absorbent particles and electrophoretic means.

14. A microfluidic device as recited in claim 10, Wherein said first sample chamber further comprises constituents for enabling amplification of said target nucleic acid.

15. A microfluidic device as recited in claim 10, wherein if said first sample portion contains at least a single molecule of said target nucleic acid, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single amplification step.

16. A microfluidic device comprising:

a first sample chamber, said first sample chamber comprising means for minimizing diffusion of said first sample portion; and

at least one amplification targeting reagent positioned in said first sample chamber,

whereby if a sample portion which comprises at least a single molecule of a target nucleic acid is positioned in said first sample chamber, said first sample portion would attain a detectable concentration of said target nucleic acid within a portion of said first sample chamber after a single round of amplification.

17. A microfluidic device as recited in claim 16, wherein said means for minimizing diffusion of said first sample portion comprises at least one physical restraint on diffusion.

18. A microfluidic device as recited in claim 16, wherein said means for minimizing diffusion of said first sample portion comprises at least one chemical restraint on diffusion.

19. A microfluidic device as recited in claim 16, wherein said means for minimizing diffusion of said first sample portion comprises at least one feature selected from among flow restriction means, glass beads, gels, absorbent particles and electrophoretic means.