Simultaneous detection of protein isoforms and nucleic acids from low starting cell numbers

Patent metadata

Field	Value
Publication number	US-12038407-B2
Application number	US-202117327806-A
Country	US
Kind code	B2
Filing date	May 24, 2021
Priority date	Dec 14, 2018
Publication date	Jul 16, 2024
Grant date	Jul 16, 2024

Abstract

Dual nucleic acid and protein isoform measurements are performed on low starting cell numbers (e.g. equivalent to the number of blastomeres composing early embryonic development stages (morula and blastocysts)), comprising integrating fractionation polyacrylamide gel electrophoresis (fPAGE) of 10-100 cells with off-chip analysis of nucleic acids in the nuclei.

First claim

The invention claimed is:

1. A polyacrylamide gel-based device that integrates electrophoretic separation of cytoplasmic proteins and extraction of nucleic acids from nuclei, the device comprising a polyacrylamide gel covalently grafted to a polyester film, wherein the gel comprises microwells and corresponding separation lanes, wherein each microwell contains fractionated nuclei of cells, and each corresponding separation lane comprises electrophoretically separated proteins of the cells.

2. The device of claim 1 , wherein each microwell abuts the corresponding separation lane.

3. The device of claim 1 , wherein each microwell is contained in an excised gel raft, wherein a void created by excision of the gel raft abuts the corresponding separation lane.

4. The device of claim 1 , wherein each microwell is contained in an excised gel raft, wherein a void created by excision of the gel raft abuts the corresponding separation lane, and each gel raft is contained in a reaction vessel wherein nucleic acid from then nuclei is extracted.

5. The device of claim 1 , wherein the proteins are photocaptured by UV-light activated benzophenone moieties incorporated in the gel.

6. The device of claim 1 wherein the gel is 100-150 μm-thick, and the microwells are cylindrical of diameters of 20-160 μm.

7. The device of claim 1 wherein each microwell contains fractionated nuclei of 1-100 cells.

8. The device of claim 1 wherein each microwell contains fractionated nuclei of 10-100 cells.

9. The device of claim 1 , further comprising a membrane comprising a transverse electrophoretic protein blot of the gel.

10. The device of claim 1 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a biopsy.

11. The device of claim 1 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

12. The device of claim 2 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

13. The device of claim 3 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

14. The device of claim 4 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

15. The device of claim 5 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

16. The device of claim 6 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

17. The device of claim 7 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

18. The device of claim 8 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

19. The device of claim 9 wherein each microwell contains fractionated nuclei of cells, wherein the cells are of a single embryo or single blastomere.

20. A method integrating electrophoretic separation of cytoplasmic proteins and extraction of nucleic acids from nuclei using the device of claim 1 , the method comprising
performing on the contained nuclei a genomic or transcriptomic measurement; and
performing on the separated proteins a protein measurement.

Assignees

• Univ California

Inventors

• Herr Amy E
• He Lin
• Modzelewski Andrew J
• Rosàs-Canyelles Elisabet

Classifications

• C08L33/26

  Homopolymers or copolymers of acrylamide or methacrylamide · CPC title

• C08K5/1545

  Six-membered rings · CPC title

• C08J2333/26

  Homopolymers or copolymers of acrylamide or methacrylamide · CPC title

• C08J9/40

  Impregnation · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title