ANALYSIS USING MICROFLUIDIC PARTITIONING DEVICES
First Claim
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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.
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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.
130 Citations
21 Claims
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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.
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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. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for conducting an analysis, comprising:
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(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. - View Dependent Claims (12, 13, 14, 15, 16)
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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. - View Dependent Claims (18, 19)
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.
- -U1-B1-F-3′ and
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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.
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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. - View Dependent Claims (21)
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Specification