Method and device for detecting the presence of a single target nucleic acid in a sample

• US 8,563,275 B2
• Filed: 08/11/2012
• Issued: 10/22/2013
• Est. Priority Date: 04/17/1997
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Inventors: Brown, James F., Silver, Jonathan E.
• Primary Examiner(s): Sisson, Bradley L
• Application Number:
  US13/572,649
• Publication Number:
  US 20120301884A1
• Time in Patent Office: 437 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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 comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.

Claims

1. A method for amplifying a target nucleic acid when present in a sample, the method comprising:

introducing to sample retaining means a single aqueous sample and a nongaseous fluid that is immiscible with the sample, wherein the sample comprises reagents sufficient to perform a primer-based amplification of a nucleic acid when present in said sample;

segregating the sample into a plurality of aliquots, wherein the aliquots have reaction volumes ranging from 1 picoliter to 1 microliter, and wherein the aliquots are segregated and entrapped by the nongaseous fluid in the sample retaining means; and

subjecting the plurality of aliquots in the sample retaining means to a primer-based amplification reaction whereby reagent primers anneal to the target nucleic acid to amplify the target nucleic acid when said target nucleic acid is present in said sample.

2. The method of claim 1, wherein the reaction volumes range from 1 picoliter to 100 nanoliters.

3. The method of claim 1, wherein the reaction volumes range from 1 picoliter to 10 nanoliters.

4. The method of claim 1, wherein the nongaseous fluid is oil.

5. The method of claim 1, wherein the reagents comprise a detectable moiety sufficient to permit detection of amplified target nucleic acid and wherein the method further comprises detecting the aliquots that yield amplified target nucleic acid from the subjecting to the primer-based amplification reaction.

6. The method of claim 1, wherein, after the subjecting, at least one of the aliquots yields amplified target nucleic acid, and at least some of the aliquots do not yield amplified target nucleic acid.

7. The method of claim 1, further comprising physically restraining the aliquots in the sample retaining means to permit local accumulation of amplified target nucleic acid when present, wherein the physical restraining is via at least one of beads, gels, and absorbent particles disposed in the sample retaining means.