Reaction system for performing in the amplification of nucleic acids

• US 7,264,950 B1
• Filed: 09/29/2000
• Issued: 09/04/2007
• Est. Priority Date: 09/29/1999
• Status: Expired due to Fees

• Current Assignee: Secretary of State For Defence
• 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: Squirrell, David James, Lee, Martin Alan, Wenn, David, Leslie, Dario Lyall, Bird, Hilary, Shaw, John, Deacon, Julie
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/089,498
• Time in Patent Office: 2,531 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• 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 an amplification reaction, said method comprising supplying to a reagent well in a disposable unit, a reaction mixture comprising:

(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, wherein the pH of said buffer system is from 8.7-9.0, 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 and a facing layer having one or more reagent wells up to 1000 microns in depth defined therebetween, and wherein said reaction mixture further comprises a detergent and/or a blocking agent.

3. The method according to claim 1 or 2, wherein the buffer system comprises from 30-70 mM Tris HCl.

4. The method according to claim 3, wherein the buffer system comprises about 50 mM Tris HCl, pH 8.8 at 25°

C.

5. The method according to claim 1 wherein the reaction mixture further comprises from 0.01 to 0.1% v/v detergents.

6. The method according to claim 1 wherein the reaction is effected in the presence of a blocking agent which comprises bovine serum albumin (BSA).

7. The method according to claim 1 wherein the thermally conducting layer of the disposable unit is metal.

8. The method according to claim 7 wherein the thermally conducting metal layer of the disposable unit is aluminum.

9. The method according to claim 7 wherein the thermally conducting metal layer is coated with a plastic or other biocompatible layer.

10. The method according to claim 9 wherein the biocompatible layer is polystyrene.

11. The method according to claim 1 wherein the thermally conducting layer and the facing layer of the disposable unit are heat-sealed together.

12. The method according to claim 1 wherein the facing layer of the disposable unit comprises a thermally conducting layer.

13. The method according to claim 1 wherein the facing layer of the disposable unit is of a transparent biocompatible plastics material.

14. The method according to claim 1, wherein the disposable unit further comprises a spacing layer having holes and channels to define reagent wells and channels adhered between the thermally conducting layer and the facing layer.

15. The method according to claim 14 wherein the layers are adhered together by means of a biocompatible adhesive and the reaction is effected in the presence of a blocking agent which comprises bovine serum albumin.

16. The method according to claim 1 wherein spacer means are provided within each reagent well.

17. The method according to claim 1 wherein the reagent wells are pre-dosed with dried reagents.

18. The method according to claim 17 wherein the dried reagents are PCR reagent primers or probes.

19. The method according to claim 1 wherein the disposable unit contains a plurality of reagent wells and each reagent well is fed by a common channel which has a single opening to the outside of the unit.

20. The method according to claim 1 wherein the disposable unit is placed in apparatus comprising at least two heating blocks, each of which is under the control of an automatic temperature control means, and conveyor means for holding and transferring a disposable unit sequentially between the blocks.

21. The method according to claim 20 wherein the apparatus further comprises an actuator above each block and arranged to clamp the disposable unit against the respective block.

22. The method according to claim 20 wherein the at least one of the heating blocks is segregated and each segment is held at a different temperature.

23. The method according to claim 1 wherein the disposable unit is integral with or arranged in close proximity to an electrically conducting polymer.

24. The method according to claim 1 wherein the presence of labelled reagents within the disposable unit is monitored.

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

(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, wherein the pH of said buffer system is at or above ¹8.8, 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 and a facing layer having one or more reagent wells up to 1000 microns in depth defined therebetween, and wherein said reaction mixture further comprises a detergent and/or a blocking agent.

25. A method of filling a disposable unit for conducting thermal cycling reactions with a liquid, comprising:

using air pressure to force liquid into the disposable unit;

wherein the disposable unit for conducting thermal cycling reactions comprises a thermally conducting layer and a facing layer having a plurality of reagent wells defined therebetween;

wherein all the reagent wells are fed by a common channel which includes a single opening to the outside of the disposable unit;

and wherein the method comprises placing the disposable unit and said liquid in a vacuum chamber, reducing pressure in said vacuum chamber such that air is evacuated from the disposable unit, immersing at least the single opening of said unit in said liquid, and increasing pressure in said vacuum chamber such that liquid is forced to enter the disposable unit through the single opening.

26. The method according to claim 25 wherein the single opening is immersed in said liquid before the pressure in the vacuum chamber is reduced.