ANALYSIS USING MICROFLUIDIC PARTITIONING DEVICES

• US 20090317798A1
• Filed: 06/02/2006
• Published: 12/24/2009
• Est. Priority Date: 06/02/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Daridon, Antoine, Heid, Christian A.
• Application Number:
  US11/916,025
• Publication Number:
  US 20090317798A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0874 Three dimensional network
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• 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 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The invention relates to methods, reagents and devices for detection and characterization of nucleic acids, cells, and other biological samples. Assay method are provided in which a sample is partitioned into sub-samples, and analysis of the contents of the sub-samples carried out. The invention also provides microfluidic devices for conducting the assay. The invention also provides an analysis method using a universal primers and probes for amplification and detection.

Claims

1. An assay method comprising:

(a) partitioning a sample obtained from the blood of a pregnant woman into a plurality of sub-samples,

wherein said sample comprises a plurality of nucleic acid molecules, and wherein most of said sub-samples comprise either (i) 0 or 1 target nucleic acid sequences or (ii) 0 or 1 cells;

(b) providing sufficient reagents in each sub-sample to amplify at least two different target sequences;

(c) amplifying target sequence(s) in the two sub-sample(s)

thereby producing amplicon(s) in the sub-sample(s);

(d) querying the sub-samples for the presence of an amplicon, or a property of an amplicon, to obtain an indication of fetal disease or propensity to disease.

18. The method of claim 1, wherein nucleic acid molecules in the sample are free in solution.

19. The method of claim 1, wherein the sample comprises fetal cells.

20. The method of claim 1, wherein the indication of fetal disease or propensity to disease comprises a genetic defect selected from the group consisting of a substitution, an amplification, a deletion, and a translocation.

21. The method of claim 1, wherein the sub-samples are queried for a polymorphism.

22. The method of claim 21, wherein the polymorphism comprises a single nucleotide polymorphism (SNP).

23. The method of claim 1, wherein the sub-samples are queried to quantify a target sequence.

24. The method of claim 1, wherein the sub-samples are queried with a fetal-specific probe.

25. The method of claim 1, wherein the sample is partitioned into at least 10⁴ sub-samples.

26. The method of claim 25, wherein each sub-sample has a volume of less than one nanoliter.

27. The method of claim 1, wherein said nucleic acid molecules comprise DNA.

28. The method of claim 27, wherein said DNA comprises cDNA made from RNA.

29. The method of claim 1, wherein sufficient reagents are provide to amplify at least 10 target sequences, if present.

30. The method of claim 1, wherein said amplification is by PCR or RT-PCR.

31. The method of claim 1, wherein the sample comprises a plurality of cells comprising nucleic acid molecules, and wherein partitioning the sample comprises partitioning intact cells into a plurality of sub-samples.

32. The method of claim 1, wherein at least 99% of the sub-samples comprise either (i) 0 or 1 target nucleic acid sequences or (ii) 0 or 1 cells.

2-17. -17. (canceled)