Reaction system for performing in the amplification of nucleic acids

• US 7,659,096 B2
• Filed: 07/30/2007
• Issued: 02/09/2010
• Est. Priority Date: 09/29/1999
• Status: Expired due to Term

• Current Assignee: The secretary of state for defence in her Britannic Majesty's Government of the United Kingdom of Great Britain And Northern Ireland
• Original Assignee: The secretary of state for defence in her Britannic Majesty's Government of the United Kingdom of Great Britain And Northern Ireland
• Inventors: Wenn, David, Leslie, Dario Lyall, Bird, Hilary, Shaw, John, Deacon, Julie, Squirrell, David James, Lee, Martin Alan
• Primary Examiner(s): Kim; Young J
• Application Number:
  US11/830,283
• Publication Number:
  US 20080176232A1
• Time in Patent Office: 925 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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 method of carrying out a rapid amplification reaction, the method comprising supplying to a reagent well in a disposable unit:

(a) a sample that contains or is suspected of containing a target nucleic acid sequence;

(b) primers, nucleotides and enzymes required to effect the amplification reaction;

(c) a buffer system; and

(d) a blocking agent, and

subjecting the disposable unit to amplification conditions such that target nucleic acid present within the sample is amplified,

wherein the disposable unit comprises a thermally conducting layer, comprising a metal layer, and a facing layer having one or more reagent wells of up to 1000 microns in depth defined therebetween.

2. The method of claim 1, wherein the amplification reaction is carried out in less than 20 minutes.

3. The method of claim 1, wherein the amplification reaction is carried out in approximately 19 minutes.

4. The method of claim 1, wherein the buffer system has a concentration of 30-70 mM, and wherein the pH of the buffer system is in excess of 8.3.

5. The method of claim 1, wherein the thermally conducting metal layer is an aluminium layer.

6. The method of claim 1, wherein the thermally conducting layer is coated with a biocompatible layer.

7. The method of claim 6, wherein the biocompatible layer is a plastic layer.

8. The method of claim 6, wherein the biocompatible layer is a polystyrene layer.

9. The method of claim 1, wherein the enzymes required to effect the amplification reaction comprise Taq DNA polymerase.

10. The method of claim 1, wherein the blocking agent comprises bovine serum albumin (BSA).

11. The method of claim 10, wherein the bovine serum albumin has a concentration of 250 ng/μ

l.

12. A method of carrying out an amplification reaction, the method comprising supplying to a reagent well in a disposable unit:

(a) a sample that contains or is suspected of containing a target nucleic acid sequence;

(b) primers, nucleotides and enzymes required to effect the amplification reaction; and

(c) a buffer system having a concentration of 30-70 mM; and

(d) a blocking agent, and

subjecting the disposable 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, comprising a metal layer, and a facing layer having one or more reagent wells of up to 1000 microns in depth defined therebetween.

13. The method of claim 12 wherein the buffer system is 50 mM.

14. The method of claim 12 wherein the buffer system additionally comprises a detergent.

15. The method of claim 12 wherein the buffer system additionally comprises (NH₄)₂SO₄.

16. The method of claim 12 wherein the buffer system additionally comprises a detergent and (NH₄)₂SO₄.

17. The method of claim 12 wherein the buffer system additionally comprises a TWEEN™

detergent and (NH₄)₂SO₄.

18. The method of claim 12 wherein the pH of the buffer system is above 8.3.

19. The method of claim 12, wherein the thermally conducting metal layer is an aluminium layer.

20. The method of claim 12, wherein the thermally conducting layer is coated with a biocompatible layer.

21. The method of claim 20, wherein the biocompatible layer is a plastic layer.

22. The method of claim 20, wherein the biocompatible layer is a polystyrene layer.

23. The method of claim 12, wherein the enzymes required to effect the amplification reaction comprise Taq DNA polymerase.

24. The method of claim 12, wherein the blocking agent comprises bovine serum albumin (BSA).

25. The method of claim 24, wherein the bovine serum albumin has a concentration of 250 ng/μ

l.