Pcr Process And Arrangement For Dna Amplification Using Dry Reagents

• US 20080241890A1
• Filed: 04/26/2005
• Published: 10/02/2008
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Siemens AG
• Original Assignee: Siemens AG
• Inventors: Paulicka, Peter, Gumbrecht, Walter, Stanzel, Manfred
• Granted Patent: US 7,993,828 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

A PCR process for DNA amplification with thermocyclisation of the corresponding reagents is disclosed, in which total integration of all substances and process steps is achieved in a closed, single-use unit (so-called cartridge) in which the reagents are stored in a storage-stable form at room temperature. According to the process, the water-soluble reagents are covered by a water-insoluble medium, then the DNA to be amplified is supplied and the water-insoluble medium is eliminated, so that the water-soluble reagents are dissolved and PCR can start. In the corresponding arrangement, a test unit designed as a single-use produce (a so-called cartridge) has at least one micro-channel or micro-cavity for receiving a PCR reagent. The PCR reagents in the form of a mixture which can be dried at a negligible vapour pressure and forms a storage-stable substance substance at room temperature adhere to the walls of the micro-channel or micro-cavity and form a thin film covered by an insoluble medium.

Claims

1. A process for DNA amplification by PCR with thermocycling of the substances containing the DNA together with the associated reagents, the process comprising:

integrating materials in a closed analysis unit including a single-use cartridge with at least one of microchannels and microcavities, in which at least the reagents necessary for the PCR are held in a form which is stable when stored at room temperature;

firstly covering the water-soluble reagents with a water-insoluble medium; and

supplying the DNA to be amplified in an aqueous solvent, wherein

the covering effect of the water-insoluble medium is subsequently negated, so that

the water-soluble reagents dissolve in the aqueous solvent,

after which the PCR thermocycling reactions can start.

2. The process as claimed in claim 1, wherein water-free substances, storable for several months at room temperature and which make it possible to carry out a PCR after supplying water, are used as reagents for the PCR.

3. The process as claimed in claim 1, wherein for DNA bound in biological structures, the disintegration of the structures, is integrated with the PCR.

4. The process as claimed in claim 1, wherein an analysis unit including a geometry that permits efficient and rapid thermocycling is used.

5. The process as claimed in claim 1, wherein a disposable product that is small and inexpensive to make in mass production is used as the analysis unit for performing the reactions.

6. The process as claimed in claim 1, wherein the PCR reactions are carried out in parallel for different DNA amplifications in an array arrangement, optionally with different primers.

7. The process as claimed in claim 6, wherein PCR reagents with different compositions are used for the different DNA amplifications.

8. The process as claimed in claim 6, wherein the array is filled with a DNA sample, wherein the lid closes the PCR cavities and isolates them fluidically from one another, wherein the water-insoluble medium is paraffin, and wherein the paraffin layers are melted and individual DNA amplifications are carried out in parallel.

30. The process as claimed in claim 2, wherein the PCR reactions are carried out in parallel for different DNA amplifications in an array arrangement, optionally with different primers.

31. The process as claimed in claim 30, wherein PCR reagents with different compositions are used for the different DNA amplifications.

32. The process as claimed in claim 30, wherein the array is filled with a DNA sample, wherein the lid closes the PCR cavities and isolates them fluidically from one another, wherein the water-insoluble medium is paraffin, and wherein the paraffin layers are melted and individual DNA amplifications are carried out in parallel.

9. An arrangement for DNA amplification by PCR with thermocycling of the substances containing the DNA together with the associated reagents, the arrangement comprising:

an analysis unit, designed as a single-use cartridge, including at least one microchannel and/or microcavity to receive a PCR reagent, wherein the PCR reagents are introduced as a mixture forming a substance that is stable at room temperature and are covered with a layer of a water-insoluble medium, supplying the DNA to be amplified in an aqueous solvent, the water-soluble reagents being coverable with a water-insoluble medium and the covering effect of the water-insoluble medium being subsequently negatable, such that the water-soluble reagents are dissolvable in the aqueous solvent, after which the PCR thermocycling reactions can start.

10. The arrangement as claimed in claim 9, wherein the mixture is a water-free solid.

11. The arrangement as claimed in claim 9, wherein the solid adheres to the walls of the microchannel or of the microcavity.

12. The arrangement as claimed in claim 9, wherein a PCR reagent film is formed by way of a film-forming agent.

13. The arrangement as claimed in claim 9, wherein the water-insoluble medium is paraffin.

14. The arrangement as claimed in claim 13, wherein the paraffin has a defined melting point for releasing the PCR reagents.

15. The arrangement as claimed in claim 9, characterized wherein a lysis reagent, including specific properties, is furthermore provided in order to disintegrate cells in the microcavity or in the microchannel.

16. The arrangement as claimed in claim 15, wherein the lysis reagent consists of dryable substances with a negligible vapor pressure, which are stable when stored at room temperature, and the lysis reagent adheres to the walls of the microchannel upstream of the PCR cavity, the channel for the lysis substance opening into the PCR cavity.

17. The arrangement as claimed in claim 9, wherein substrates which have DNA-binding properties are provided in the microchannel or in the microcavity.

18. The arrangement as claimed in claim 17, wherein the substrates are DNA-binding magnet beads.

19. The arrangement as claimed in claim 18, wherein the lysis reagents and the magnet beads are contained in a common dry matrix.

20. The arrangement as claimed in claim 9, wherein an input port or whole blood samples is provided.

21. The arrangement as claimed in claim 9, wherein means for supplying water are provided.

22. The arrangement as claimed in claim 9, wherein means are provided for mixing at least one of a blood sample and water, and a buffer solution.

23. The arrangement as claimed in claim 9, wherein means are provided so that at least one of blood, blood/water and blood/buffer mixtures can flow through the microchannel or microcavity coated with lysis/bead reagent.

24. The arrangement as claimed in claim 9, wherein means are provided for generating a magnetic field for the purpose of fixing a DNA-magnet bead complex in the PCR cavity.

25. The arrangement as claimed in claim 9, wherein thermocycling means are provided.

26. The arrangement as claimed in claim 9, wherein means are provided for at least partial pressure equilibration in the PCR cavity.

27. The arrangement as claimed in claim 9, wherein an array of PCR cavities is provided.

28. The arrangement as claimed in claim 9, wherein different PCR reagents are provided in the PCR cavities.

29. The arrangement as claimed in claim 9, wherein a lid is provided for closing the PCR cavities during the PCR.