Apparatus and methods for parallel processing of micro-volume liquid reactions

• US 20020072096A1
• Filed: 02/20/2001
• Published: 06/13/2002
• Est. Priority Date: 02/18/2000
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: OʼKeefe, Matthew, Foreman, Pamela K.
• Granted Patent: US 7,332,271 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01F 2215/0431 Numerical size values, e.g. diameter of a hole or conduit, area, volume, length, width, or ratios thereof
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/30351 using hydrophilic/hydrophobic surfaces
• CPC Class Codes: B01F 35/712 for feeding fluids
• CPC Class Codes: B01F 35/7172 using capillary forces
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/0074 Biological products
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/10 Means to control humidity and/or other gases
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/50857 using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: B01L 3/5088 : confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: H01J 49/0418 : for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials

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.

Claims

1. A method for simultaneously conducting a plurality of micro-volume polynucleotide amplification reactions, the method comprising:

(a) introducing a plurality of liquid samples into the sample chambers of a microhole apparatus, wherein the samples contain necessary polynucleotide amplification reaction components; and

(b) placing the apparatus into an environment favorable to the polynucleotide amplification reaction;

wherein the microhole apparatus comprises a substrate, wherein the substrate 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 a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber; and

wherein the apparatus is substantially free of contaminating amplifiable polynucleotides.

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 1 wherein the polynucleotide amplification reaction is a ligase chain reaction or a rolling circle amplification reaction.

5. The method according to claim 1, wherein results of the reactions are monitored.

6. The method according to claim 5 wherein the results are monitored by a method selected from the group consisting of optical monitoring, mass spectrometry and electrophoresis.

7. The method according to claim 1, wherein progress of the reactions are monitored during the course of the reactions.

8. 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.

9. The method according to claim 1, wherein a reagent is affixed to the substrate.

10. The method according to claim 9 wherein the environment is selected from the group consisting of a hydrophobic medium and a humidified chamber.

11. The method according to claim 1, wherein the analysis is used to detect a genetic polymorphism.

12. The method according to claim 9, wherein the analysis is used to detect a genetic polymorphism.

13. The method according to claim 1, wherein the analysis is used to analyze gene expression levels.

14. The method according to claim 9, wherein the analysis is used to analyze gene expression levels.

16. An apparatus according to claim 15 wherein the apparatus comprises at least two reagents used in a polynucleotide amplification reaction to be carried out in the apparatus.

17. An apparatus according to claim 15 wherein the sample chamber has a height to width ratio of about 1:

1, wherein the height of the sample chamber is measured from one face of the substrate to the other.

18. An apparatus according to claim 15 wherein the substrate comprises hydrophobic regions, wherein the hydrophobic regions are located on the substrate such that a liquid sample present in one sample chamber does not intermix with a liquid sample present in another sample chamber.

19. An apparatus according to claim 18, wherein the substrate comprises an upper face and a lower face.

20. An apparatus according to claim 19, wherein the through axes of the sample chambers are perpendicular to both faces of the substrate.

21. An apparatus according to claim 20, wherein the sample chamber has the shape of a right circular cylinder.

22. An apparatus according to claim 20, wherein the sample chamber has the shape of a right polygonal prism.

23. An apparatus according to claim 18, wherein the hydrophobic regions are located on the upper and lower faces of the substrate such that the openings of at least one sample chamber from at least one adjacent sample chamber by a hydrophobic region.

24. An apparatus according to claim 23, wherein additional hydrophobic regions are located on the walls of the sample chambers.

25. An apparatus according to claim 18, wherein hydrophobic regions are located on the walls of the sample chambers.

26. An apparatus according to claim 25, wherein the hydrophobic region forms an annular ring along the wall of the sample chamber.

27. An apparatus according to claim 25, 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.

28. An apparatus according to claim 15, wherein the at least one reagent is affixed to the substrate.

29. An apparatus according to claim 15, wherein the polynucleotide amplification reaction is a polymerase chain reaction.

30. An apparatus according to claim 15, wherein the polynucleotide amplification reaction is a ligase chain reaction or a rolling circle amplification reaction.

15. An apparatus 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 one or more walls and an opening at each end, and

(c) holds a sample such that the sample is retained in the apparatus 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 apparatus is substantially free of contaminating amplifiable polynucleotides; and

wherein the apparatus comprises at least one reagent used in a polynucleotide amplification reaction to be carried out in the apparatus.

31. A kit comprising an apparatus for containing multiple micro-volume liquid samples for performing a polynucleotide amplification reaction, comprising a substrate, wherein the substrate defines a plurality of sample chambers, wherein each sample chamber:

(a) extends through the substrate,

(b) comprises one or more walls and an opening at each end, and

(c) holds a sample such that the sample is retained in the apparatus 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 apparatus is substantially free of contaminating amplifiable polynucleotides;

and further comprising a polynucleotide amplification reaction component packaged in a suitable container.

32. A kit according to claim 31, wherein the polynucleotide amplification reaction component is affixed to the substrate.

33. A kit according to claim 31, wherein the kit further comprises a hydrophobic substance to be used with the apparatus.

34. A kit according to claim 33, wherein the hydrophobic substance is a hydrophobic fluid packaged in a suitable container.

35. A kit according to claim 33, wherein the hydrophobic substance is a hydrophobic cover.

36. A kit according to claim 31, further comprising a chamber for maintaining the appropriate environmental conditions for a polynucleotide amplification reaction to be carried out in the apparatus.

37. A kit according to claim 31, further comprising a device for loading samples into the sample chambers.