Solid gel amplification method and apparatus for genotyping and pathogen detection

• US 8,993,270 B2
• Filed: 07/08/2011
• Issued: 03/31/2015
• Est. Priority Date: 07/09/2010
• Status: Active Grant

• Current Assignee: The Governors of the University of Alberta (University of Alberta)
• Original Assignee: The Governors of the University of Alberta (University of Alberta)
• Inventors: Atrazhev, Alexey, Acker, Jason
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/809,106
• Publication Number:
  US 20130149709A1
• Time in Patent Office: 1,362 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention provides for a novel system and method for amplification and detection of nucleic acids within a miniaturized device.

Claims

1. A method for detecting a nucleic acid molecule within a hydrogel post array comprising

a) providing an array of hydrogel posts, each hydrogel post containing a respective cell-free, enzymatic, nucleic-acid amplification system, at least one of the respective amplification systems being different from at least another of the respective amplification systems;

b) applying nucleic acid molecules to at least one of said hydrogel posts, at least one of the nucleic acid molecules comprising a template for said amplification system; and

c) incubating said hydrogel posts under conditions promoting the synthesis of an amplified nucleic acid product by said amplification system from said at least one template.

2. The method of claim 1 wherein the hydrogel posts contain a marker, wherein said marker has different properties, capable of being measured, when interacting with double-stranded nucleic acids than with single-stranded nucleic acids.

3. The method of claim 2 wherein said measurement is in real-time and the marker is a fluorescent marker.

4. The method of claim 3 wherein said hydrogel post is comprised of polyacrylamide of 2.0-12% weight per unit volume, and photopolymerized in the absence of ammonium persulfate (APS).

5. The method of claim 1 wherein said template is included in said hydrogel posts.

6. The method of claim 1 wherein said template is provided externally to said hydrogel posts.

7. The method of claim 1 wherein the template is between 3,800 and 250,000,000 base pairs in size.

22. The method of claim 1 wherein said amplification systems comprise at least two non-immobilized nucleic acids, each of the at least two non-immobilized nucleic acids being capable of promoting synthesis of amplified nucleic acid product from said template.

8. A method for detecting a nucleic acid molecule within a hydrogel post array comprising

a) providing an array of hydrogel posts, each hydrogel post containing a cell-free, enzymatic, nucleic-acid amplification system, at least one of the respective amplification systems being different from at least another of the respective amplification systems;

b) including nucleic acid molecules in at least one of said hydrogel posts, at least one of which may comprise a template for said amplification system; and

c) incubating said hydrogel posts under conditions promoting the synthesis of an amplified nucleic acid product by said amplification system from said at least one template.

9. The method of claim 8 wherein the hydrogel posts contain a marker, wherein said marker has different properties, capable of being measured, when interacting with double-stranded nucleic acids than with single-stranded nucleic acids.

10. The method of claim 9 wherein the marker is a fluorescent marker.

11. The method of claim 10 wherein said hydrogel post is comprised of polyacrylamide of 2.0-12% weight per unit volume, and photopolymerized in the absence of ammonium persulfate (APS).

12. The method of claim 8 wherein said template is included in said hydrogel posts.

13. The method of claim 8 wherein said template is provided externally to said hydrogel posts.

14. The method of claim 8 wherein the template is between 3,800 and 250,000,000 base pairs in size.

23. The method of claim 8 wherein said amplification systems comprise at least two non-immobilized nucleic acids, each of the at least two non-immobilized nucleic acids capable of promoting synthesis of amplified nucleic acid product from said template.

15. A method for detecting a nucleic acid molecule within a hydrogel post array comprising

a) providing an array of hydrogel posts, each hydrogel post containing a respective cell-free, enzymatic, nucleic-acid amplification system, at least one of the respective amplification systems being different from at least another of the respective amplification systems;

b) introducing nucleic acid molecules in a solution in fluid communication with at least one of said hydrogel posts, at least one of which may comprise a template for said amplification system; and

c) incubating said hydrogel posts under conditions promoting the synthesis of an amplified nucleic acid product by said amplification system from said at least one template.

16. The method of claim 15 wherein the hydrogel posts contain a marker, wherein said marker has different properties, capable of being measured, when interacting with double-stranded nucleic acids than with single-stranded nucleic acids.

17. The method of claim 16 wherein the marker is a fluorescent marker.

18. The method of claim 17 wherein said hydrogel post is comprised of polyacrylamide of 2.0-12% weight per unit volume, and photopolymerized in the absence of ammonium persulfate (APS).

19. The method of claim 15 wherein said template is included in said hydrogel posts.

20. The method of claim 15 wherein said template is provided externally to said hydrogel posts.

21. The method of claim 15 wherein the template is between 3,800 and 250,000,000 base pairs in size.

24. The method of claim 15 wherein said amplification systems comprise at least two non-immobilized nucleic acids, each of the at least two non-immobilized nucleic acids capable of promoting synthesis of amplified nucleic acid product from said template.