DNA isolation method

• US 6,852,851 B1
• Filed: 10/20/2000
• Issued: 02/08/2005
• Est. Priority Date: 10/28/1999
• Status: Expired due to Fees

• Current Assignee: Gyros Patent AB (Mesa Laboratories Inc.)
• Original Assignee: Gyros AB (Mesa Laboratories Inc.)
• Inventors: Kenrick, Michael Kenneth, Tooke, Nigel Eric, Thomas, Philip Landeg
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US10/111,822
• Time in Patent Office: 1,572 Days
• Field of Search: 435/306.1, 435/317.1, 536/25.4
• US Class Current: 536/25.4
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Abstract

Disclosed is a method and apparatus for the isolation of DNA or cell nuclei or a mixture thereof from cell samples in a CD device. The method includes treating a suspension of whole cells with a lysis reagent so as to lyse the cytoplasmic membrane and at least some of the nuclear membranes, and introducing the lysate into micro-channels of a microfabricated apparatus in which each of the micro-channels is provided with a barrier disposed in the channel to impede the passage or flow of DNA and cell nuclei each while allowing the passage of liquid through the micro-channel so that a mesh comprising DNA is formed in the channel.

Claims

1. A method of isolating DNA and/or cell nuclei in a microfabricated apparatus that contains a plurality of radially dispersed micro-channels the method comprising forming a mesh from a cell sample in one or more of the micro-channels which mesh acts as a barrier to DNA and/or cell nuclei.

2. The method according to claim 1 wherein the mesh formed in the said one or more micro-channels of the microfabricated apparatus comprises a mesh of nucleic acid.

3. A method of isolating DNA or cell nuclei or a mixture thereof from cells, which method comprises:

a) treating a suspension of whole cells with a lysis reagent so as to lyse the cytoplasmic membranes and at least some of the nuclear membranes;

b) introducing the lysate from step a) into micro-channels of a microfabricated apparatus wherein each of said micro-channels incorporates means to impede the passage or flow of DNA and cell nuclei while allowing the passage of liquid through the micro-channel whereby a mesh comprising DNA is formed in the channel; and

,

c) washing the mesh comprising DNA.

4. The method according to claim 3 wherein the step a) a proportion of the nuclear membranes is left intact.

5. The method according to claim 3, wherein the microfabricated apparatus comprises a rotatable disc, the disc comprising a sample introduction port located towards the center of the disc in contact with an annular sample reservoir which in turn is connected to a plurality of radially dispersed micro-channels, each micro-channel comprising an inlet channel and an outlet channel disposed towards the circumferential edge of the disc and means incorporated in each of the micro-channels to impede the passage or flow of DNA and cell nuclei, while allowing the passage of liquid therethrough.

6. The method according to claim 3, wherein said means incorporated in each of the micro-channels to impede the passage or flow of DNA and cell nuclei comprises a barrier.

7. The method according to claim 6 wherein said barrier comprises beads that impede the flow or passage of DNA and cell nuclei in the micro-channel while allowing the passage of liquid.

8. The method according to claim 7 wherein said beads are selected from the group consisting of polystyrene beads, styrene acrylate co-polymer beads, polystyrene cross-linked with divinylbenzene beads, polyvinyltoluene beads, polymethacrylate beads, polycarbonate beads, polysaccharide beads, metal oxides beads, glass beads and carbon beads.

9. The method according to claim 6 wherein said barrier comprises raised structures disposed in the micro-channel.

10. The method according to claim 9 wherein said raised structures are disposed on the base portion of the micro-channel and are moulded to form pillars such that they impede the flow or passage of DNA and cell nuclei in the micro-channel while allowing the passage of liquid.

11. The method according to claim 3, wherein at least some of the micro-channels of the microfabricated apparatus each further comprise a reaction chamber for performing assays or processing DNA.

12. The method according to claim 3, wherein the whole cells of step a) are selected from plant cells and animal cells.

13. The method according to claim 3, wherein step c) is performed by passing a washing solution through the mesh formed in the micro-channel.

14. The method according to claim 3, wherein cell nuclei captured by the mesh are further treated to release DNA.

15. A microfabricated apparatus for isolating DNA or cell nuclei or a mixture thereof from cells, which apparatus comprises a rotatable disc, the disc comprising a sample introduction port located towards the center of the disc in contact with an annular sample reservoir which in turn is connected to a plurality of radially dispersed micro-channels, each micro-channel comprising an inlet channel and an outlet channel disposed towards the circumferential edge of the disc and a barrier disposed in each of the micro-channels to impede the passage or flow of DNA and cell nuclei, while allowing the passage of liquid there through, said barrier being selected from beads or raised structures.

16. The apparatus according to claim 15 wherein said raised structures are disposed on the base portion of the micro-channel and are moulded to form pillars.