Method and assembly for DNA isolation with dry reagents

• US 8,088,576 B2
• Filed: 04/28/2005
• Issued: 01/03/2012
• Est. Priority Date: 04/30/2004
• Status: Active Grant

• Current Assignee: Siemens AG
• Original Assignee: Siemens AG
• Inventors: Gumbrecht, Walter, Paulicka, Peter, Stanzel, Manfred
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US11/587,581
• Publication Number:
  US 20070219366A1
• Time in Patent Office: 2,441 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/082 Handling hazardous material
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• 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: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label

Abstract

A DNA isolation method by removal of constituents, interfering with a subsequent PCR is disclosed. According to an embodiment of the method, all substances and method steps are fully integrated into a closed unit (cartridge) for single use, which allows entry of a DNA-containing sample and DNA-binding substrates are used for isolating the released DNA. In particular, when the method is applied to DNA isolation from whole blood by disruption of white blood cells, the reagents, required for carrying out the cell disruption and other reactions, are stored in a form which is stable at room temperature. For disrupting the white blood cells and for DNA isolation, a dry stored lysis reagent is dissolved in an aqueous solution and brought into contact with the white blood cells. The corresponding assembly includes a unit, for housing DNA-containing biological containers and/or reagents, whereby at least one microchannel is provided to contain reagents, whereby the reagent is present in the microchannel as a dry mixture with a negligible vapor pressure, which forms a stable substance at room temperature.

Claims

1. A method for DNA isolation while removing constituents that interfere with a subsequent PCR, the method comprising:

integrating materials in a closed analysis single-use unit which allows a sample with DNA to enter;

releasing the DNA by using dry reagents stored in the unit in a form that is stable at room temperature;

isolating the released DNA using DNA-binding substrates, wherein the DNA-binding substrates are magnetic beads;

bringing a dry matrix, lying on a wall in a disintegration channel of the unit and including lysis reagents and the DNA-binding substrates, in contact with biological structures to disintegrate the biological structures containing the DNA, the magnetic beads being distributed in the dry matrix such that the DNAs released by contact with the lysis reagents are bound directly and simultaneously to the DNA-binding substrates; and

collecting the substrates with the bound DNAs and transporting the collected substrates to a site of the PCR.

2. The method as claimed in claim 1, wherein white blood cells are used as the biological structures containing DNA.

3. The method as claimed in claim 2, wherein the lysis reagents are brought in contact with the white blood cells with the aid of water, the lysis reagents being stored dry and storage-stable.

4. The method as claimed in claim 1, further comprising diluting the sample with DNA, dissolving or suspending the dry reagents, disintegrating the biological structures and binding the DNA released by contact with the lysis reagents in a single step, after the sample with DNA is in the unit.

5. The method as claimed in claim 1, wherein the released DNA is bound and transported by the magnetic beads to at least one of a collection site and a reaction site.

6. The method as claimed in claim 1, wherein the magnetic beads are concentrated by applying a magnetic field.

7. The method as claimed in claim 6, wherein flowing magnetic beads are concentrated by a static magnetic field.

8. The method as claimed in claim 1, wherein the PCR is carried out by thermocycling with dryly-stored, water-soluble reagents at a reaction site.

9. The method as claimed in claim 1, wherein the sample with DNA is a blood sample put into the unit diluted and pumped through a reagent channel to a reaction channel for the PCR.

10. The method as claimed in claim 9, wherein residual blood, blood constituents and the reagent waste remain in the unit after the DNA isolation, so that a user cannot directly contact the residual blood, the blood constituents and the reagent waste.

11. The method as claimed in claim 1, wherein a disposable product is used as the single-use unit in which the PCR is performed.

12. The method as claimed in claim 11, wherein the disposable product is a cartridge.

13. The method as claimed in claim 12, wherein the cartridge is made of plastic.

14. The method as claimed in claim 1, wherein the dry matrix, having the magnetic beads therein, is adhered to the wall by adding a film-forming agent to the dry matrix.