Apparatus and methods for parallel processing of micro-volume liquid reactions
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Abstract
Disclosed herein are apparatuses and methods for conducting multiple simultaneous micro-volume chemical and biochemical reactions in an array format. In one embodiment, the format comprises an array of microholes in a substrate. Besides serving as an ordered array of sample chambers allowing the performance of multiple parallel reactions, the arrays can be used for reagent storage and transfer, library display, reagent synthesis, assembly of multiple identical reactions, dilution and desalting. Use of the arrays facilitates optical analysis of reactions, and allows optical analysis to be conducted in real time. Included within the invention are kits comprising a microhole apparatus and a reaction component of the method(s) to be carried out in the apparatus.
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Citations
63 Claims
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1-37. -37. (canceled)
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38. A microhole device for containing multiple micro-volume liquid samples consisting essentially of a substrate, wherein the substrate is solid and defines a plurality of sample chambers, wherein each sample chamber:
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(a) extends through the substrate;
(b) comprises a wall and an opening at each end;
(c) comprises at least one PCR primer reversibly affixed to said wall; and
(d) holds a sample such that the sample is retained in the sample chamber through surface tension and such that (i) a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber, and (ii) the liquid samples are maintained in the sample chambers when the microhole device is submerged in a hydrophobic medium. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47)
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48. A kit comprising a microhole device for containing multiple micro-volume liquid samples for performing a polynucleotide amplification reaction, said microhole device consisting essentially of a substrate, wherein the substrate is solid and defines a plurality of sample chambers, wherein each sample chamber:
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(a) extends through the substrate;
(b) comprises a wall and an opening at each end;
(c) comprises at least one PCR primer reversibly affixed to said wall; and
(d) holds a sample such that the sample is retained in the sample chamber through surface tension and such that (i) a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber, and (ii) the liquid samples are maintained in the sample chambers when the microhole device is submerged in a hydrophobic medium;
the kit further comprising;
(i) a polynucleotide amplification reaction component packaged in a suitable container or (ii) a hydrophobic substance to be used with the microhole device. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions, the method comprising:
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(a) introducing a plurality of liquid samples into the sample chambers of a microhole apparatus, whereby the chambers contain necessary polynucleotide amplification reaction components;
(b) protecting the samples from evaporation with a hydrophobic medium; and
(c) placing the apparatus into an environment favorable to the polynucleotide amplification reaction;
wherein the microhole apparatus comprises a substrate, wherein the substrate is solid and defines a plurality of sample chambers, wherein each sample chamber;
(i) extends through the substrate;
(ii) comprises one or more walls and an opening at each end; and
(iii) holds a sample such that the sample is retained in the apparatus through surface tension and such that (i) a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber and (ii) the liquid samples are maintained in the sample chambers when the microhole device is submerged in a hydrophobic medium for a polynucleotide amplification reaction.
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61. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions, the method comprising:
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(a) introducing a plurality of liquid samples into the sample chambers of a microhole apparatus, whereby the chambers contain necessary polynucleotide amplification reaction components;
(b) protecting the samples from evaporation; and
(c) placing the apparatus into an environment favorable to the polynucleotide amplification reaction;
wherein the microhole apparatus comprises a substrate, wherein the substrate is solid and defines a plurality of sample chambers, wherein each sample chamber;
(i) extends through the substrate;
(ii) comprises one or more walls and an opening at each end; and
(iii) holds a sample such that the sample is retained in the apparatus through surface tension and such that (i) a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber and (ii) the liquid samples are maintained in the sample chambers when the microhole device is submerged in a hydrophobic medium for a polynucleotide amplification reaction. - View Dependent Claims (62, 63)
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Specification