ANALYSIS USING MICROFLUIDIC PARTITIONING DEVICES

• US 20110143949A1
• Filed: 11/12/2010
• Published: 06/16/2011
• 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:
  US12/945,506
• Publication Number:
  US 20110143949A1
• Time in Patent Office: Days
• US Class Current: 506/7
• 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 into a plurality of sub-samples,

wherein said sample comprises a plurality of nucleic acid molecules, and wherein at least two sub-samples comprise at least one nucleic acid molecule;

(b) providing sufficient reagents in each sub-sample to amplify a target sequence or sequences;

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

thereby producing amplicons in the sub-sample;

(d) distributing the amplicons into a plurality of aliquots; and

,

(e) for each aliquot, determining a property of amplicons in the aliquot.

2. An assay method comprising

(a) partitioning a sample into a plurality of sub-samples,

wherein said sample comprises a plurality of nucleic acid molecules, and wherein at least two sub-samples comprise at least one nucleic acid molecule;

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

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

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

(d) combining the amplicons from said at least two sub-samples to create an amplicon pool;

(d) dividing the amplicon pool into a plurality of aliquots; and

,

(e) for each aliquot, determining a property of amplicons in the aliquot.

3. The method of claim 2 wherein the sample is partitioned into at least 10⁴ sub-samples.

4. The method of claim 3 wherein each subsample has a volume of less than 1 nanoliter.

5. The method of claim 2 wherein said nucleic acid molecules comprise RNA.

6. The method of claim 2 wherein sufficient reagents are provided to amplify at least 10, 20, or 50 different target sequences, if present.

7. The method of claim 2 wherein said amplification is by PCR or RT-PCR.

8. The method of claim 2 wherein the amplicon pool is divided into at least 10, 20, 50 or 100 aliquiots.

9. The method of claim 2 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.

10. The method of claim 2 wherein the sample comprises only one cell.

11. A method for conducting an analysis, comprising:

(a) partitioning a sample comprising a plurality of separable cells into at least 1000 separate reaction chambers in a MPD, wherein after partitioning at least two reaction chambers each comprise exactly one cell;

(b) providing in each reaction chamber one or more reagents for determining a property or properties of a cell, wherein the same reagents are provided in each chamber; and

(c) determining at least two different properties of a single cell in a chamber and/or determining at least one property for at least two different cells in different chambers.

12. The method of claim 11 wherein at least 99% of the reaction chambers contain zero or one cell.

13. The method of claim 11 wherein the cells are bacterial cells.

14. The method of claim 11 wherein said reagents comprise reagents for nucleic acid amplification.

15. The method of claim 11 wherein at least one property is the presence or absence in the cell of a nucleic acid having a specified sequence.

16. The method of claim 11 wherein at least one property is other than the presence or absence in the cell of a nucleic acid having a specified sequence.

17. A method for amplification and detection of multiple target DNA sequences in a sample, said method comprising

a) providing a sample containing

i) multiple target DNA sequences,

ii) a primer pair corresponding to each of said multiple target DNA sequences, each pair consisting of a first primer comprising U₁, B₁ and F domains in the order 5′

-U₁-B₁-F-3′ and

a second primer comprising U₂ and R domains in the order 5′

-U₂-R-3′

, wherein each pair of F and R primers is capable of annealing specifically to a different target DNA sequence under stringent annealing conditions;

iii) a universal primer pair capable of amplifying a double stranded DNA molecule with the structure

5′

U₁-----U₂′

-3′

3′

U₁′

-----U₂-3′

where U₁′

is the sequence complementary to U₁ and U₂′

is the sequence complementary to U₂;

b) subjecting the sample to multiple cycles of melting, reannealing, and DNA synthesis thereby producing amplicons for each of said multiple target DNA sequences, and

c) detecting the amplicons using a probe that anneals to sequence of the amplicon having the sequence of the B₁ domain or its complement.

18. The method of claim 17

wherein the sample also contains a second set of multiple target sequences, a primer pair corresponding to to each of the target sequences in the second set, each pair consisting of a first primer comprising U₁, B₂ and F domains in the order 5′

-U₁-B₂-F-3′ and

a second primer comprising U₂ and R domains in the order 5′

-U₂-R-3′

, wherein each pair of F and R oligonucleotides is capable of annealing specifically to a different target DNA sequence in the second set of multiple target sequences under stringent annealing conditions; and

wherein amplicons for each of the multiple target DNA sequences of the second set are produced; and

detecting the amplicons for each of the multiple target DNA sequences using a probe that anneals to sequence of the amplicon having the sequence of the B₂ domain or its complement.

19. The method of claim 17 wherein U₁, B₁, F₁, U₂ and R₁ domains are between 6 and 25 nucleotides in length and the probe is a molecular beacon.

20. A microfluidic device, comprising

(a) a first region comprising

(i) a flow channel formed within an elastomeric material and having a first end and a second end in fluid communication with each other through said channel, wherein said channel may be branched or unbranched;

(ii) an inlet for introducing a sample fluid in communication with said channel, said inlet;

(iii) an outlet in communication with said flow channel;

(iv) a plurality of control channels overlaying the flow channel(s),

wherein an elastomeric membrane separates the control channels from the flow channels at each intersection, the elastomeric membrane disposed to be deflected into or withdrawn from the flow channel in response to an actuation force, and

wherein, when the control channels are actuated the flow channel is partitioned into at least 1000 reaction chambers not in fluidic communication with each other;

(b) a second region compromising

a channel or chamber interposed between and in communication with said outlet in (a) and a flow channel in the third region;

(c) a third region comprising a plurality of flow channels in fluidic communication with the channel or chamber of the second region, with a region of each flow channel defining a reaction site;

(d) a control channel or channels that when actuated separates the first and second regions;

(e) a control channel or channels that when actuated separates the second and third regions; and

(f) a control channel or channels that when actuated separates the reaction sites of said flow channels from the other portions of control channels.

21. The device of claim 20 wherein the flow channels in the third region are blind flow channels.