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

• US 7,972,778 B2
• Filed: 03/11/2004
• Issued: 07/05/2011
• 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: Silver, Jonathan E., Brown, James F.
• Primary Examiner(s): Chin; Christopher L
• Application Number:
  US10/798,857
• Publication Number:
  US 20040171055A1
• Time in Patent Office: 2,672 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 a target nucleic acid molecule in a sample, said method comprising:

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

loading the sample into a sample chamber comprising a flow through channel, said sample chamber including a device for retaining a reaction product of amplification of the target nucleic acid;

subjecting the sample in said sample chamber to a homogeneous amplification assay comprising at least a first round of amplification under conditions such that amplification of said target nucleic acid occurs and a reaction product of the amplification of a single molecule of the target nucleic acid comprises a clonal amplified product having a detectable concentration within a portion of said sample chamber after a single round of amplification; and

detecting the clonal amplified product of said target nucleic acid after said first round of amplification.

2. A method as recited in claim 1, wherein said sample has a volume of about 1 picoliter or less.

3. A method as recited in claim 1, wherein said sample has a volume of about 1 nanoliter or less.

4. A method as recited in claim 1, wherein said sample has a volume of about 10 nanoliters or less.

5. A method as recited in claim 1, wherein said sample has a volume of about 100 nanoliters or less.

6. A method as recited in claim 1, wherein said sample has a volume of about 1 microliter or less.

7. A method as recited in claim 1, wherein said sample has a volume which is nanoliter-sized.

8. A method for detecting a target nucleic acid molecule in a sample, said method comprising:

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

loading the sample into a sample chamber comprising a flow through channel, said sample chamber including means for retaining a reaction product of amplification of the target nucleic acid;

subjecting the sample to a homogeneous amplification assay comprising at least a first round of amplification under conditions such that amplification of said target nucleic acid occurs and a reaction product of the amplification of a single molecule of the target nucleic acid comprises a clonal amplified product having a detectable concentration within a portion of said sample chamber after a single round of amplification; and

detecting the clonal amplified product of said target nucleic acid after said first round of amplification.

9. A method as recited in claim 8, wherein said sample has a volume of about 1 picoliter or less.

10. A method as recited in claim 8, wherein said sample has a volume of about 1 nanoliter or less.

11. A method as recited in claim 8, wherein said sample has a volume of about 10 nanoliters or less.

12. A method as recited in claim 8, wherein said sample has a volume of about 100 nanoliters or less.

13. A method as recited in claim 8, wherein said sample has a volume of about 1 microliter or less.

14. A method as recited in claim 8, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 100 microns or less.

15. A method as recited in claim 8, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 500 microns or less.

16. A method as recited in claim 8, wherein said sample chamber is positioned in a device which comprises a plurality of sample chambers.

17. A method for detecting a target nucleic acid molecule in a sample, said method comprising:

loading a sample into a sample chamber comprising a flow through channel, said sample chamber comprising constituents for enabling amplification of a target nucleic acid, said sample chamber including a device for retaining a reaction product of amplification of the target nucleic acid;

subjecting the sample in said sample chamber to a homogeneous amplification assay comprising at least a first round of amplification under conditions such that amplification of said target nucleic acid occurs and a reaction product of the amplification of a single molecule of the target nucleic acid comprises a clonal amplified product having a detectable concentration within a portion of said sample chamber after a single round of amplification; and

detecting the clonal amplified product of said target nucleic acid after said first round of amplification.

18. A method as recited in claim 17, wherein said sample has a volume of about 1 picoliter or less.

19. A method as recited in claim 17, wherein said sample has a volume of about 1 nanoliter or less.

20. A method as recited in claim 17, wherein said sample has a volume of about 10 nanoliters or less.

21. A method as recited in claim 17, wherein said sample has a volume of about 100 nanoliters or less.

22. A method as recited in claim 17, wherein said sample has a volume of about 1 microliter or less.

23. A method as recited in claim 17, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 100 microns or less.

24. A method as recited in claim 17, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 500 microns or less.

25. A method as recited in claim 17, wherein said sample chamber is positioned in a device which comprises a plurality of sample chambers.

26. A method for detecting a target nucleic acid molecule in a sample, said method comprising:

loading a sample into a sample chamber comprising a flow through channel, said sample chamber comprising constituents for enabling amplification of a target nucleic acid, said sample chamber including means for retaining a reaction product of amplification of the target nucleic acid;

subjecting the sample to a homogeneous amplification assay comprising at least a first round of amplification under conditions such that amplification of said target nucleic acid occurs and a reaction product of the amplification of a single molecule of the target nucleic acid comprises a clonal amplified product having a detectable concentration within a portion of said sample chamber after a single round of amplification; and

detecting the clonal amplified product of said target nucleic acid after said first round of amplification.

27. A method as recited in claim 26, wherein said sample has a volume of about 1 picoliter or less.

28. A method as recited in claim 26, wherein said sample has a volume of about 1 nanoliter or less.

29. A method as recited in claim 26, wherein said sample has a volume of about 10 nanoliters or less.

30. A method as recited in claim 26, wherein said sample has a volume of about 100 nanoliters or less.

31. A method as recited in claim 26, wherein said sample has a volume of about 1 microliter or less.

32. A method as recited in claim 26, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 100 microns or less.

33. A method as recited in claim 26, wherein said sample chamber is confined in at least one dimension by opposing barriers separated by a dimension of about 500 microns or less.

34. A method as recited in claim 26, wherein said sample chamber is positioned in a device which comprises a plurality of sample chambers.

35. A method for detecting a target nucleic acid molecule in a sample, said method comprising:

loading a sample into a sample chamber comprising a flow through channel, said sample chamber further comprising constituents for cloning a segment of DNA, said sample chamber including a device for retaining a reaction product from cloning the segment of DNA;

subjecting the sample in said sample chamber to a homogeneous amplification assay comprising at least a first round of amplification under conditions such that amplification of said segment of DNA occurs and a reaction product of the amplification of a single molecule of the target nucleic acid comprises the cloned DNA segment product having a detectable concentration within a portion of said sample chamber after a single round of amplification; and

detecting the cloned DNA segment after said first round of amplification.