PCR process and arrangement for DNA amplification using dry reagents

• US 7,993,828 B2
• Filed: 04/26/2005
• Issued: 08/09/2011
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Siemens AG
• Original Assignee: Siemens AG
• Inventors: Paulicka, Peter, Gumbrecht, Walter, Stanzel, Manfred
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US11/587,567
• Publication Number:
  US 20080241890A1
• Time in Patent Office: 2,296 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 substances containing the DNA together with associated water-soluble 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 water-soluble reagents necessary for the PCR are held in a form which is stable when stored at room temperature, and in which the water-soluble reagents are collectively arranged in a layer;

providing a covering layer, consisting essentially of a water-insoluble medium, in the single-use cartridge, wherein the water-insoluble medium is paraffin;

covering the layer of water-soluble reagents with the covering layer;

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; and

performing PCR thermocycling reactions on the aqueous solvent including the DNA to be amplified.

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, disintegration of the biological structures is integrated with the PCR.

4. The process as claimed in claim 1, wherein an analysis unit including a compact cartridge geometry is used.

5. The process as claimed in claim 1, wherein a disposable product 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 a lid closes the microcavities and isolates them fluidically from one another, and wherein layers of the paraffin are melted and individual DNA amplifications are carried out in parallel.

9. 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.

10. The process as claimed in claim 9, wherein PCR reagents with different compositions are used for the different DNA amplifications.

11. The process as claimed in claim 9, wherein the array is filled with a DNA sample, wherein a lid closes the microcavities and isolates them fluidically from one another, and wherein layers of the paraffin are melted and individual DNA amplifications are carried out in parallel.

12. The process as claimed in claim 1, wherein the water-soluble reagents are stored in a solid form.

13. The process as claimed in claim 1, wherein a single boundary interface is formed between the covering layer and the layer of the water-soluble reagents.

14. The process as claimed in claim 1, wherein some of the water-soluble reagents are on a surface of the layer of water-soluble reagents, and are interposed between others of the water-soluble reagents within the layer of water-soluble reagents and the covering layer.

15. The process as claimed in claim 1, wherein the covering layer, consisting essentially of a water-insoluble medium, is provided in one of the microcavities of the single-use cartridge.