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

• US 9,518,299 B2
• Filed: 12/02/2014
• Issued: 12/13/2016
• Est. Priority Date: 02/18/2000
• Status: Expired due to Term

• 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: O'Keefe, Matthew T., Foreman, Pamela K.
• Primary Examiner(s): Beisner, William H
• Application Number:
  US14/558,501
• Publication Number:
  US 20150087550A1
• Time in Patent Office: 742 Days
• US Class Current: 1/1
• 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. A method for simultaneously conducting a plurality of polynucleotide reactions, the method comprising:

introducing a plurality of liquid samples comprising a nucleic acid into a plurality of holes of a substrate having a first surface and a second surface and the plurality of holes extending through the substrate from the first surface to the second surface;

coating openings of the plurality of holes on the first and second surfaces of the substrate with a hydrophobic fluid; and

simultaneously conducting a plurality of polynucleotide reactions on the plurality in at least some of the plurality of holes.

2. The method of claim 1, wherein the polynucleotide reaction is a polynucleotide amplification reaction.

3. The method of claim 1, further comprising sealing the plurality of holes with an evaporation barrier.

4. The method of claim 3, wherein the evaporation barrier comprises a silicone rubber sheet.

5. The method of claim 3, wherein the evaporation barrier comprises a polymeric or glass cover slide.

6. The method of claim 5, wherein the cover slide is optically clear.

7. The method of claim 6, further comprising optically monitoring progress of the reactions in the plurality of holes.

8. The method of claim 7, wherein optically monitoring further comprises monitoring by fluorescence spectroscopy.

9. The method of claim 1, wherein each hole or set of holes is circumscribed by a narrow raised feature.

10. The method of claim 1, further comprising supplementing the reactions with one or more reagents during the course of the reactions.

11. The method of claim 1, further comprising affixing a target nucleic acid to a wall of a hole by surface tension or desiccation.

12. The method of claim 1, wherein the plurality of liquid samples is obtained from a single sample comprising the target nucleic acid sequence.

13. The method of claim 12 wherein the single sample is a biological sample.

14. The method of claim 1, wherein the substrate comprises a sequence-specific polynucleotide amplification reaction component reversibly affixed to a wall of a hole.

15. The method of claim 14, wherein the sequence-specific polynucleotide amplification reaction component is dissolved when the liquid sample is introduced into the hole.

16. The method of claim 1, wherein the liquid samples are terminally diluted to the extent that an average number of the holes contain exactly one copy of the target nucleic acid.

17. The method of claim 1, wherein haplotype information for a target nucleic acid sequence is generated.

18. The method of claim 17, wherein a sequence-specific probe is used to detect a genetic polymorphism.