Apparatus and methods for parallel processing of microvolume liquid reactions

• US 10,227,644 B2
• Filed: 04/27/2018
• Issued: 03/12/2019
• Est. Priority Date: 02/18/2000
• Status: Expired due to Fees

• 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: O'Keefe, Matthew T., Foreman, Pamela K.
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US15/965,155
• Publication Number:
  US 20180245133A1
• Time in Patent Office: 319 Days
• 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 system for conducting a plurality of polynucleotide reactions, the system comprising:

a substrate comprising an upper face, a lower face, and a plurality of holes disposed between the upper face and the lower face;

a dispenser that dispenses (1) liquid samples comprising nucleic acids, (2) reagents, and (3) oil into the plurality of holes;

a bath comprising an oil, the lower face of the substrate being submerged in the oil;

a thermal cycler that performs amplification reactions on the liquid samples dispensed within the plurality of holes to produce amplified products in the plurality of holes.

2. The system of claim 1, wherein the plurality of holes comprise a plurality of through-holes in the substrate.

3. The system of claim 1, further comprising an evaporation barrier that seals the plurality of holes.

4. The system of claim 1, wherein the dispenser dispenses the samples in the plurality of holes via the upper face of the substrate.

5. The system of claim 1, wherein the system creates sample libraries within the plurality of holes of the substrate.

6. The system of claim 1, wherein the lower face of the substrate is immersed in the oil and the oil coats the samples.

7. The system of claim 1, wherein the the lower face of the substrate is immersed in the oil to submerge, overlay, or coat the samples with the oil.

8. The system of claim 1, wherein the thermal cycler comprises the bath.

9. The system of claim 1, wherein the thermal cycler thermally cycles at least one of the oil or the bath.

10. The system of claim 1, wherein the oil comprises mineral oil or silicone oil.

11. The system of claim 1, wherein the substrate comprises a plate.

12. The system of claim 1, wherein the thermal cycler comprises a heat block in thermal contact with the substrate and thermally cycles the liquid samples with the heat block.

13. The system of claim 1, further comprising a sequencer, wherein the system produces an amplified product for loading into the sequencer.

14. The system of claim 1, wherein the dispenser is a pipette.

15. A system for conducting a plurality of polynucleotide reactions, the system comprising:

a substrate comprising an upper face, a lower face, and a plurality of holes disposed between the upper face and the lower face;

a dispenser that dispenses (1) liquid samples comprising nucleic acids and (2) reagents into the plurality of holes, and a pipette that dispenses oil into the plurality of holes, the dispenser including an apparatus comprising samples arranged in a pattern matching a pattern of the plurality of holes;

a bath comprising an oil, the lower face of the substrate being submerged in the oil; and

a thermal cycler that performs amplification reactions on the liquid samples dispensed within the plurality of holes to produce amplified products in the plurality of holes.

16. The system of claim 15, wherein the lower face of the substrate is immersed in the oil to submerge, overlay, or coat the samples with the oil.

17. The system of claim 15, wherein the oil comprises mineral oil or silicone oil.

18. The system of claim 15, wherein the substrate comprises a plate.

19. The system of claim 15, further comprising a sequencer, wherein the system produces an amplified product for loading into the sequencer.

20. The system of claim 15, wherein first dispenser is a first pipette and the second dispenser is a second pipette.