Reaction system for performing in the amplification of nucleic acids

• US 8,986,927 B2
• Filed: 09/03/2009
• Issued: 03/24/2015
• Est. Priority Date: 09/29/1999
• Status: Active Grant

• Current Assignee: Secretary of State For Defence
• Original Assignee: Secretary of State For Defence
• Inventors: Lee, Martin Alan, Bird, Hilary, Leslie, Dario Lyall, Squirrell, David James, Shaw, John, Wenn, David, Deacon, Julie
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/553,367
• Publication Number:
  US 20090325278A1
• Time in Patent Office: 2,028 Days
• US Class Current: 435/6.1
• CPC Class Codes: B01L 3/52 Containers specially adapted for storing or dispensing a reagent B01L3/02 takes precedence; containers for medical or pharmaceutical purposes A61J1/00; containers in general B65D; storing or dispensing test elements G01N33/4875; automated reagent dispensing G01N35/1002
• CPC Class Codes: B01L 3/523 with means for closing or opening
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046

Abstract

A method of carrying out an amplification reaction, said method comprising supplying to a well in a disposable unit (a) a sample which contains or is suspected of containing a target nucleic acid sequence (b) primers, nucleotides and enzymes required to effect said amplification reaction and (c) a buffer system, and subjecting the unit to thermal cycling conditions such that any target nucleic acid present within the sample is amplified; wherein the disposable unit comprises a thermally conducting layer and a facing layer having one or more reagent wells of up to 1000 microns in depth defined therebetween; and the reaction mixture comprises at least one of the following: A) a buffer system wherein the pH is above 8.3; B) a detergent; and/or C) a blocking agent. Apparatus for effecting the method as well as disposable units for use in the method are described. The method is particularly suitable for rapid PCR reactions.

Claims

1. A disposable unit comprising a thermally conducting layer and a facing layer separated by a spacing layer having multiple reagent wells defined therein, the reagent wells having well openings through which sample is introduced, and wherein the well openings of the reagent wells and one or both of the thermally conducting layer and the facing layer of the unit are configured such that once the reagent wells are filled with sample, the well openings of the reagent wells are sealable by mechanically deforming one or both of the thermally conducting layer and the facing layer of the unit, without heat-sealing.

2. The disposable unit of claim 1, wherein at least some reagent wells are pre-dosed with dried reagents.

3. The disposable unit of claim 2, wherein the dried reagents are PCR reagent primers or probes.

4. The disposable unit of claim 2, wherein the pre-dosed reagent wells are pre dosed with a different PCR primer.

5. The disposable unit of claim 2, wherein the pre-dosed reagent wells are pre dosed with polymerase.

6. The disposable unit of claim 1, wherein the spacing layer comprises holes and channels defining the one or more reagent wells and one or more channels.

7. The disposable unit of claim 1, wherein the layers of the disposable unit are secured by adhesive.

8. The disposable unit of claim 1 wherein multiple reagent wells are fed by one or more channels and each of the multiple reagent wells has a single opening onto one of the one or more channels.

9. The disposable unit of claim 1 wherein all the reagent wells are fed by a common channel which includes a single opening to outside of the unit.

10. The disposable unit of claim 1, wherein the thermally conducting layer comprises a metal.

11. The disposable unit of claim 10, wherein the metal comprises aluminum.

12. The disposable unit of claim 1, wherein the one or more reagent wells are 100-1000 microns in depth.

13. The disposable unit of claim 1, wherein the one or more reagent wells are 100-500 microns in depth.

14. The disposable unit if claim 1, wherein the facing layer is transparent.

15. The disposable unit of claim 1, wherein the disposable unit is used for conducting nucleic acid amplification, wherein the spacing layer has a thickness less than 500 μ

m, and wherein the reagent wells are left unsealed to allow reagent materials to be introduced, and subsequently closed by mechanically deforming the thermally conducting layer and the facing layer.

16. A disposable unit comprising a thermally conducting layer and a facing layer separated by a spacing layer having multiple reagent wells of up to 500 microns in depth defined therein, wherein each well is sealable by mechanically deforming one or both of the thermally conducting layer and the facing layer once filled.

17. The disposable unit of claim 16, wherein the spacing layer comprises holes and channels defining the multiple reagent wells and one or more channels.