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