초록 ▼

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 per

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.

대표청구항 ▼

What is claimed is: 1. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions in a microhole device, the method comprising: (a) providing a microhole device having a substrate, wherein the substrate is solid and defines a plurality of sample chambe

What is claimed is: 1. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions in a microhole device, the method comprising: (a) providing a microhole device having a substrate, wherein the substrate is solid and defines a plurality of sample chambers for retaining liquid samples through surface tension wherein each sample chamber comprises a wall and an opening at each end such that a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber, each sample chamber containing at least one desired polynucleotide amplification reaction component, wherein such component having the possibility of being distinct from a reaction component in a different sample chamber; (b) introducing a liquid sample containing at least one additional desired polynucleotide amplification reaction component into the sample chambers of the microhole device; (c) submerging the device in a hydrophobic medium; and (d) placing the submerged device into any environment favorable to the polynucleotide amplification reaction. 2. The method according to claim 1 wherein the environment is selected from the group consisting of a hydrophobic medium and a humidified chamber. 3. The method according to claim 1 wherein the polynucleotide amplification reaction is a polymerase chain reaction. 4. The method according to claim 3, wherein the desired polynucleotide amplification reaction component is affixed to the wall of said sample chamber by surface tension or desiccation. 5. The method according to claim 4 wherein the desired polynucleotide amplification reaction component is a PCR primer. 6. The method according to claim 3, wherein the polymerase chain reaction is used to detect a genetic polymorphism. 7. The method according to claim 3, wherein the polymerase chain reaction is used to analyze gene expression levels. 8. The method according to claim 1, wherein the results of the reactions are monitored by mass spectrometry or electrophoresis. 9. The method according to claim 1, wherein the progress of the reactions are monitored by optical monitoring during the course of the reactions. 10. The method according to claim 1, wherein one or more of the reactions are supplemented with one or more reagents during the course of the reaction. 11. The method according to claim 1 wherein the microhole device consists essentially of a substrate and the substrate is titanium. 12. The method according to claim 1, wherein the microhole device consists essentially of a substrate and the substrate comprises top and bottom surfaces that each contain raised features which form closed curves circumscribing the openings to said sample chambers. 13. The method of claim 1, wherein each sample chamber has a height to width ratio of less than or equal to 2:1. 14. A method according to claim 1, wherein a plurality of sample chambers holds a distinct sample. 15. 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: (a) extends through the substrate; (b) comprises a wall and an opening at each end; (c) comprises a hydrophobic annular ring on the wall of the chamber, separating hydrophobic regions; (d) comprises at least one PCR primer reversibly affixed to said wall; and (e) 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. 16. A microhole device according to claim 15 wherein the sample chamber comprises at least two PCR primers used in a polynucleotide amplification reaction to be carried out in the microhole device. 17. A microhole device according to claim 15, wherein the substrate comprises an upper face and a lower face and the through axes of the sample chambers are perpendicular to both faces of the substrate. 18. A microhole device according to claim 17, wherein the sample chamber has the shape of a right circular cylinder. 19. A microhole device according to claim 17, wherein the sample chamber has the shape of a right polygonal prism. 20. A microhole device according to claim 17, wherein hydrophobic regions are located on the upper and lower faces of the substrate such that the openings of at least one sample chamber are separated from at least one adjacent sample chamber by a hydrophobic region. 21. A microhole device according to claim 17, comprising two or more hydrophobic regions, each forming an annular ring along the wall of the sample chamber, wherein the hydrophobic regions define one or more annular non-hydrophobic rings therebetween. 22. A microhole device according to claim 15, comprising additional hydrophobic regions located on the walls of the sample chambers. 23. A microhole device according to claim 15, wherein the at least one PCR primer is affixed to the sample chamber of the microhole device by desiccation. 24. A microhole device according to claim 15, wherein substrate is titanium. 25. A microhole device according to claim 15 wherein the substrate comprises top and bottom surfaces that each contain raised features which form closed curves circumscribing the openings to said sample chambers. 26. A microhole device for containing multiple micro-volume liquid samples comprising a substrate, wherein the substrate defines a plurality of sample chambers, wherein each sample chamber: (a) extends through the substrate; (b) comprises a wall and an opening at each end; (c) comprises a hydrophobic annular ring on the wall of the chamber, separating two hydrophilic regions; and (d) holds a sample such that the sample is retained in the sample chamber through surface tension and such that a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber. 27. A microhole device for containing multiple micro-volume liquid samples consisting essentially of a substrate, wherein the substrate defines a plurality of sample chambers, wherein each sample chamber: (a) extends through the substrate; (b) comprises a wall and an opening at each end; (c) comprises a hydrophobic annular ring on the wall of the chamber, separating two hydrophilic regions; and (d) holds a sample such that the sample is retained in the sample chamber through surface tension and such that a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber, wherein the sample chamber is substantially free of contaminating amplifiable polynucleotides, and wherein the sample chamber comprises at least one reagent used in a polynucleotide amplification reaction reversibly affixed to the wall thereof. 28. A method for simultaneously conducting a plurality of distinct sequence-specific micro-volume polynucleotide amplification reactions in a microhole device, the method comprising: (a) providing a microhole device having a substrate, wherein the substrate is solid and defines a plurality of sample chambers for retaining liquid samples through surface tension wherein each sample chamber comprises a wall and an opening at each end 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, a plurality of sample chambers containing at least one distinct desired sequence-specific amplification reaction component reversibly affixed to the wall of said sample chamber thereof; (b) introducing a liquid sample containing at least one additional polynucleotide amplification reaction component, thereby dissolving the reversibly affixed distinct sequence-specific polynucleotide amplification reaction component in the liquid samples; (c) submerging the microhole device in a hydrophobic medium; and (d) placing the submerged microhole device into any environment favorable to the distinct polynucleotide amplification reactions. 29. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions on a plurality of distinct liquid samples, the method comprising: (a) providing a microhole device having a substrate, wherein the substrate is solid and defines a plurality of sample chambers for retaining distinct liquid samples through surface tension such that a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber, each sample chamber containing at least one sequence-specific polynucleotide amplification reaction component reversibly affixed to the wall of said sample chamber; (b) introducing a distinct liquid sample that is distinct from a liquid sample in a different sample chamber into a plurality of sample chambers of the microhole device, thereby dissolving the reversibly affixed sequence-specific polynucleotide amplification reaction component in the distinct liquid sample; (c) submerging the microhole device in a hydrophobic medium; and (d) placing the submerged device into any environment favorable to the polynucleotide amplification reactions.