Device and method for amplifying and detecting target nucleic acids

• US 5,645,801 A
• Filed: 08/18/1995
• Issued: 07/08/1997
• Est. Priority Date: 10/21/1993
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Coules, Ronald A., Shain, Eric B., Gordon, Julian, Solomon, Natalie A., Bouma, Stanley R., Zaun, Peter
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/516,728
• Time in Patent Office: 690 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/283.1, 435/287.1, 435/287.2, 435/287.3, 435/288.1, 435/288.2, 435/288.7, 435/289.1, 435/290.1, 435/290.4 , 435/293.1, 435/304.1, 435/304.2, 435/810, 422/50, 422/52, 422/55, 422/57, 422/58, 422/61, 422/63, 422/68.1, 422/69, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78, 935/88
• US Class Current: 422/68.1
• 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
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods, devices, apparatus and kits for amplifying and detecting nucleic acid are provided. The apparatus is a thermal cycling device that operates in conjunction with a reaction/detection unit. A sample is loaded into a reaction chamber of the device which is then sealably mated with a detection chamber to form a sealed reaction/detection unit that is virtually irreversibly closed. One or more heating elements of the thermal cycling apparatus applies a desired temperature to the reaction/detection device to amplify target nucleic acid in the sample. The reaction mixture is then transferred to the detection chamber and amplified target nucleic acid is immobilized on a support in the detection chamber. A detection system associated with the apparatus detects and analyzes the immobilized amplified nucleic acid target. Kits include the reaction/detection units and reagents for amplification.

Claims

1. An article of manufacture for use as a nucleic acid sample analysis device comprising:

an elongated reaction unit having a lower closed end and an upper open end, said reaction unit having an interior chamber, the upper end being adapted for receiving an amplification reaction sample into the interior chamber;

a detection unit having an opening into a detection chamber, said detection chamber housing means for collecting amplified target nucleic acid, whereby said amplified target nucleic acid can enter said detection unit through said opening;

wherein at least one of said reaction unit and said detection unit includes means for sealably engaging the open end of said reaction unit to the opening of said detection unit to form a sealed reaction/detection unit such that the interior chamber of said reaction unit is in fluid communication with the detection chamber.

2. The article of claim 1 wherein said reaction unit comprises a capillary tube or a microsyringe tube closed at one end.

3. The article of claim 1 wherein the lower portion of the interior chamber of said reaction unit includes surface irregularities of the type that can be obtained by melting said lower portion.

4. The article of claim 1 wherein said means for collecting comprises a support to which are immobilized capture molecules for immobilizing target nucleic acid.

5. The article of claim 4 wherein said support comprises a porous support.

6. The article of claim 1 wherein said means for sealably engaging comprises a friction seal.

7. The article of claim 1 wherein said means for sealably engaging comprises a snap-fit.

8. The article of claim 1 wherein said means for sealably engaging comprises an irreversible lock fit.

9. The article of claim 1 wherein said detection unit includes a reservoir at a lower end and said opening opens to the detection chamber above said reservoir.

10. The article of claim 1 wherein said reaction chamber houses a propellant disposed in said reaction chamber.

11. The article of claim 10 wherein said propellant is an expandable fluid.

12. The article of claim 10 wherein said propellant is air or a reagent solution.

13. The article of claim 1 wherein at least one of said reaction unit and said detection unit includes key means on said unit for engaging a key groove in a unit holder to ensure a predetermined orientation for said unit when it is supported by said holder.

35. A kit for amplifying and detecting target nucleic acid, the kit comprising:

multiple disposable reaction/detection units of the type specified in claim 1; and

one or more containers holding in suitable buffer(s);

a DNA polymerase;

dATP, dCTP, dTTP, and dGTP; and

at least two primers specific for amplifying a predetermined target nucleic acid by the polymerase chain reaction.

36. The kit of claim 35 wherein said DNA polymerase is thermostable.

37. The kit of claim 35 wherein at least one of said primers is covalently attached to a hapten.

38. A kit for amplifying and detecting target nucleic acid, the kit comprising:

multiple disposable reaction/detection units of the type specified in claim 1; and

one or more containers holding in suitable buffer(s);

a DNA ligase;

NAD; and

at least four probes specific for amplifying a predetermined target nucleic acid by the ligase chain reaction.

39. The kit of claim 38 wherein said one or more containers further holds a polymerase and at least one deoxynucleotide triphosphate.

40. The kit of claim 38 wherein said ligase is thermostable.

41. The kit of claim 38 wherein at least one of said probes is covalently attached to a hapten.

14. An article of manufacture for use as a nucleic acid sample analysis device comprising:

an elongated reaction unit having a lower closed end and an upper open end, said reaction unit having an interior chamber, the upper end being adapted for receiving an amplification reaction sample into the interior chamber, wherein the interior houses a propellant near the closed end;

a detection unit having an opening into a detection chamber, said detection chamber housing means for collecting amplified nucleic acid, whereby said amplified target nucleic acid can enter said detection unit through said opening;

wherein at least one of said reaction unit and said detection unit includes means for sealably engaging the open end of said reaction unit to the opening of said detection unit to form a sealed reaction/detection unit such that the interior chamber of said reaction unit is in fluid communication with the detection chamber.

15. The article of claim 14 wherein said reaction unit comprises a capillary tube or a microsyringe tube closed at one end.

16. The article of claim 14 wherein the lower portion of the interior chamber of said reaction unit includes surface irregularities of the type that can be obtained by melting said lower portion.

17. The article of claim 14 wherein said means for collecting comprises a support to which is immobilized capture molecules for immobilizing target nucleic acid.

18. The article of claim 17 wherein said support comprises a porous support.

19. The article of claim 14 wherein said means for sealably engaging comprises a friction seal.

20. The article of claim 14 wherein said means for sealably engaging comprises an irreversible lock fit.

21. The article of claim 14 wherein said detection unit includes a reservoir at a lower end and said opening opens to the detection chamber above said reservoir.

22. The article of claim 14 wherein at least one of said reaction unit and said detection unit includes key means on said unit for engaging a key groove in a unit holder to ensure a predetermined orientation for said unit when it is supported by said holder.

23. A method of amplifying and detecting a target nucleic acid, comprising the steps:

(a) inserting a reaction sample into a sample analysis device comprising;

an elongated reaction unit having a lower closed end and an upper open end, said reaction unit having an interior chamber, the upper end being adapted for receiving an amplification reaction sample into the interior chamber;

a detection unit having an opening into a detection chamber, said detection chamber housing means for collecting amplified target nucleic acid, whereby said amplified target nucleic acid can enter said detection unit through said opening;

wherein at least one of said reaction unit and said detection unit includes means for sealably engaging the open end of said reaction unit to the opening of said detection unit to form a sealed reaction/detection unit such that the interior chamber of said reaction unit is in fluid communication with the detection chamber, and wherein detectable reagents for detecting said amplified target nucleic acid are disposed in said reaction/detection unit;

(b) engaging said reaction chamber and said detection chamber together to form a sealed reaction/detection unit;

(c) conducting an amplification reaction in said reaction chamber of said sealed unit;

(d) transferring the amplified reaction sample from said reaction chamber to said detection chamber of said sealed unit without disengaging the reaction unit from the detection unit; and

(e) examining said means for collecting amplified target nucleic acid for the presence of detectable reagent to determine the presence of said target nucleic acid analyte.

24. The method of claim 23 wherein said means for collecting comprises a support to which is attached at least one capture molecule for immobilizing amplified target nucleic acid.

25. The method of claim 24 wherein said immobilized amplified target nucleic acid is detected by a detectable label.

26. The method of claim 25 wherein said detectable label comprises a colloidal particle.

27. The method of claim 24 wherein said support comprises a porous support.

28. The method of claim 23 wherein said amplification reaction comprises a ligase chain reaction or a polymerase chain reaction.

29. The method of claim 23 wherein said transfer is caused by increasing the temperature of at least a portion of said reaction chamber.

30. The method of claim 23 wherein a propellant is disposed in said interior portion of the reaction chamber and said transfer comprises inducing said propellant to expand.

31. The method of claim 30 wherein said expansion is by vaporization.

32. The method of claim 31 wherein said vaporization is initiated at or near the lower closed end of the reaction chamber.

33. The method of claim 30 wherein said propellant is air.

34. The method of claim 30 wherein said fluid is the reaction sample itself.