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

Patent metadata

Field	Value
Publication number	US-9518299-B2
Application number	US-201414558501-A
Country	US
Kind code	B2
Filing date	Dec 2, 2014
Priority date	Feb 18, 2000
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

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.

First claim

What is claimed:

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.

Assignees

• Univ Leland Stanford Junior

Inventors

• O'Keefe Matthew T
• Foreman Pamela K

Classifications

• B01L3/5025 Primary

  for parallel transport of multiple samples · CPC title

• B01L3/50857

  using arrays or bundles of open capillaries for holding samples · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• B01F13/0059

  Operations & Transport · mapped topic

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title