APPARATUS AND METHODS FOR PARALLEL PROCESSING OF MICRO-VOLUME LIQUID REACTIONS

• US 20100075330A1
• Filed: 10/20/2009
• Published: 03/25/2010
• Est. Priority Date: 02/18/2000
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Foreman, Pamela K., O'Keefe, Matthew
• Granted Patent: US 8,053,185 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 35/71 Feed mechanisms with proportioning B01F35/80
• 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: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/172 : Haplotypes
• 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

1-32. -32. (canceled)

33. A method for simultaneously conducting a plurality of sequence-specific micro-volume polynucleotide amplification reactions to provide molecular haplotype information for a target nucleic acid sequence, the method comprising:

(a) introducing a plurality of liquid samples comprising a target nucleic acid into the sample chambers of a microhole apparatus, wherein the liquid samples are terminally diluted, such that on average, some of the sample chambers contain exactly one copy of the target nucleic acid, and wherein the chambers contain necessary polynucleotide amplification reaction components;

(b) protecting the samples from evaporation with a hydrophobic medium; and

(c) simultaneously conducting a plurality of sequence-specific micro-volume polynucleotide amplification reactions on the plurality of liquid samples,

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 apparatus is submerged in a hydrophobic medium.

34. The method according to claim 33, wherein a sequence-specific probe is used to detect a genetic polymorphism.

35. The method according to claim 33, wherein step (c) further comprises placing the apparatus into an environment favorable to the polynucleotide amplification.

36. The method according to claim 35, wherein the environment is selected from the group consisting of a hydrophobic medium and a humidified chamber.

37. The method of claim 33, wherein the liquid samples are maintained in the sample chambers when the microhole apparatus is submerged in a hydrophobic medium for conducting the polynucleotide amplification reaction.

38. The method according to claim 33, wherein the polynucleotide amplification reaction is a polymerase chain reaction.

39. The method according to claim 38, wherein the polymerase chain reaction is used to detect a genetic polymorphism.

40. The method according to claim 33, further comprising optically monitoring progress of the reactions in the chambers.

41. The method according to claim 40, wherein optically monitoring further comprises monitoring by fluorescence spectroscopy.

42. The method according to claim 33, further comprising supplementing the reactions with one or more reagents during the course of the reactions.

43. The method according to claim 42, further comprising affixing a target nucleic acid to a wall of a sample chamber by surface tension or desiccation.

44. The method according to claim 33, wherein the plurality of liquid samples is obtained from a single sample comprising the target nucleic acid sequence.

45. The method according to claim 44, wherein the single sample is a biological sample.

46. The method according to claim 33, wherein the microhole apparatus contains a sequence-specific polynucleotide amplification reaction component reversibly affixed to a wall of a sample chamber.

47. The method according to claim 46, wherein the sequence-specific polynucleotide amplification reaction component is dissolved when the liquid sample is introduced into the sample chamber.

48. A method of determining haplotype information for a target nucleic acid sequence, the method comprising:

(a) introducing a plurality of liquid samples comprising a target nucleic acid into the sample chambers of a microhole apparatus, wherein the liquid samples are terminally diluted, such that on average, some of the sample chambers contain exactly one copy of the target nucleic acid, and wherein the chambers contain necessary polynucleotide amplification reaction components;

(b) protecting the samples from evaporation with a hydrophobic medium;

(c) simultaneously conducting a plurality of sequence-specific micro-volume polynucleotide amplification reactions on the plurality of liquid samples; and

(d) analyzing the sequence-specific micro-volume polynucleotide amplification reactions to determine haplotype information for the target nucleic acid sequence,

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 apparatus is submerged in a hydrophobic medium.

49. The method according to claim 48, wherein a sequence-specific probe is used to detect a genetic polymorphism.

50. The method according to claim 48, wherein step (c) further comprises placing the apparatus into an environment favorable to the polynucleotide amplification.

51. The method according to claim 50, wherein the environment is selected from the group consisting of a hydrophobic medium and a humidified chamber.

52. The method of claim 48, wherein the liquid samples are maintained in the sample chambers when the microhole apparatus is submerged in a hydrophobic medium for conducting the polynucleotide amplification reaction.

53. The method according to claim 48, wherein the polynucleotide amplification reaction is a polymerase chain reaction.

54. The method according to claim 53, wherein the polymerase chain reaction is used to detect a genetic polymorphism.

55. The method according to claim 48, further comprising optically monitoring progress of the reactions in the chambers.

56. The method according to claim 55, wherein optically monitoring further comprises monitoring by fluorescence spectroscopy.

57. The method according to claim 48, further comprising supplementing the reactions with one or more reagents during the course of the reactions.

58. The method according to claim 57, further comprising affixing a target nucleic acid to a wall of a sample chamber by surface tension or desiccation.

59. The method according to claim 48, wherein the plurality of liquid samples is obtained from a single sample comprising the target nucleic acid sequence.

60. The method according to claim 59, wherein the single sample is a biological sample.

61. The method according to claim 48, wherein the microhole apparatus contains a sequence-specific polynucleotide amplification reaction component reversibly affixed to a wall of a sample chamber.

62. The method according to claim 61, wherein the sequence-specific polynucleotide amplification reaction component is dissolved when the liquid sample is introduced into the sample chamber.