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

• US 8,257,925 B2
• Filed: 05/16/2011
• Issued: 09/04/2012
• Est. Priority Date: 04/17/1997
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), The United States of America As Represented By The Secretary of Agriculture
• Inventors: Brown, James F., Silver, Jonathan E.
• Primary Examiner(s): Chin, Chris L
• Application Number:
  US13/108,959
• Publication Number:
  US 20110311979A1
• Time in Patent Office: 477 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 miniaturized assembly is provided whereby a fluid sample can be divided into a plurality of sample portions in retaining wells and the sample fluid can be displaced from open ends of the wells while simultaneously being sealed in the wells. A method of dividing a fluid sample using the assembly is also provided.

Claims

1. A method for detecting the presence of a single target nucleic acid molecule in a sample, said method comprising:

providing a sample containing at least one target nucleic acid molecule to be amplified and constituents for enabling amplification of the target nucleic acid molecule;

loading the sample into a sample chamber, said sample chamber retaining a reaction product of amplification of a single target nucleic acid molecule of said sample such that a reaction product of the amplification of the single target nucleic acid molecule attains a detectable concentration within a portion of said sample chamber after a single round of amplification when subjected to a homogeneous amplification assay;

subjecting the sample in said sample chamber to a homogeneous amplification assay including a single round of amplification under conditions such that amplification of said at least one target nucleic acid molecule occurs and the reaction product of the amplification of a single target nucleic acid molecule of said sample attains a detectable concentration within said portion of said sample chamber after said single round of amplification; and

detecting the reaction product of said single target nucleic acid molecule after said single round of amplification.

2. The method of claim 1, wherein the detecting comprises detecting fluorescence associated with the reaction product.

3. The method of claim 1, wherein the detecting comprises detecting light intensity indicative of 5′

exonuclease activity.

4. The method of claim 1, further comprising determining an initial concentration of the target nucleic acid molecule in the sample.

5. The method of claim 1, wherein the reaction product within the portion of the sample chamber is entrapped by oil.

6. The method of claim 1, wherein the detecting comprises performing fluorescence-based detection of the reaction product in a portion of a capillary tube.

7. The method of claim 6, wherein the at least one capillary tube has an inner diameter of about 500 μ

m or less.

8. The method of claim 6, wherein the at least one capillary tube has an inner diameter between about 500 μ

m to 20 μ

m.

9. The method of claim 1, further comprising protecting the reaction product from evaporation.

10. The method of claim 1, further comprising protecting the reaction product from contamination.

11. The method of claim 1, further comprising terminally diluting the sample comprises terminally diluting the sample.

12. The method of claim 1, further comprising quantifying a number of reaction products having a detectable concentration of the sample.

13. The method of claim 1, wherein the single round of amplification comprises 30 to 40 thermal cycles.

14. The method of claim 1, wherein the portion of the sample chamber is defined by a reaction volume subjected to the single round of amplification and retaining the reaction product.

15. The method of claim 14, wherein the reaction volume ranges from about 1 picoliter to about 100 nanoliters.